Equol-containing extract, method for production thereof, method for extraction of equol, and equol-containing food

ABSTRACT

The present invention relates to an extract obtained by extracting useful components containing equol from an equol-containing fermented soybean hypocotyl, and to a method for producing the same. 
     The present invention makes it possible to efficiently obtain useful components containing equol from the fermented soybean hypocotyl by subjecting an equol-containing fermented soybean hypocotyl to extraction using an ethanol aqueous solution as an extractant. The present invention reduces the content of saponin, which causes an unpleasant taste, by sequentially subjecting the equol-containing fermented soybean hypocotyl to extraction using an ethanol aqueous solution and ethanol, while efficiently extracting equol and glycitein.

TECHNICAL FIELD

The present invention relates to an equol-containing extract obtained byextracting effective components from an equol-containing fermentedsoybean hypocotyl and a production method thereof. The present inventionalso relates to a method for efficiently purifying an equol-containingsubstance to obtain a highly pure equol. The present invention furtherrelates to an equol-containing food material and an equol-containingfood.

BACKGROUND ART

The isoflavones (soybean isoflavones: daidzein, genistein, glycitein)contained in soybeans have structures similar to estradiol, and haveanti-estrogen actions associated with binding to estrogen receptors(hereinafter referred to as ER) and estrogen-like actions. The everconducted epidemiological studies and intervention studies of soybeanisoflavones suggest that they have preventive effects due to theiranti-estrogen actions on breast cancer, prostate cancer and otherhormone-dependent cancers and improving effects due to theirestrogen-like actions on menopausal disorders, postmenopausalosteoporosis and hyperlipidemia.

Recently, it has been pointed out that the active component of thephysiological effects of these soybean isoflavones may be a metaboliteof daidzein, i.e., equol. More specifically, it has been reported thatequol has an ability to bind to ER (especially to ERβ) greater thansoybean isoflavones and that it has a remarkably high transitioncapability to target organs such as breast and prostate tissues(Non-Patent Documents 1 to 4). Moreover, a case-control study reportsthat there are significantly less patients who produce equol in thepatients of breast cancer and prostate cancer. The effects of soybeanisoflavones that improve bone density and lipid metabolism were examinedregarding postmenopausal women categorized into two groups: those whoproduce equol and those who do not. A significant improvement in thosewho produce equol was observed.

Equol is produced by metabolism of daidzein by enteric bacteria. Theabilities to produce equol vary between individuals, and the percentageof Japanese who produce equol is reportedly about 50%. That is, about50% of Japanese are not able to produce equol (non-equol-producingindividuals). Such an individual cannot enjoy any useful physiologicalbenefits based on the action of equol even if they ingest soybeans andprocessed soybean foods. Therefore, in order to attain usefulphysiological benefits based on the action of equol in anon-equol-producing individual, ingesting equol itself is thought to beeffective.

Non-patent Document 1: Morito K, Hirose T, Kinjo J, Hirakawa T, Okawa M,Nohara T, Ogawa S, Inoue S, Muramatsu M, Masamune Y. Interaction ofphytoestrogens with estrogen receptors α and β. Biol Pharm Bull 24(4):351-356, 2001

Non-patent Document 2: Maubach J, Bracke M E, Heyerick A, Depypere H T,Serreyn R F, Mareel M M, Keukeleire D D. Quantitation of soy-derivedphytoestrogens in human breast tissue and biological fluids byhigh-performance liquid chromatography. J Chromatography B 784: 137-144,2003

Non-patent Document 3: Morton M S, Chan P S F, Cheng C, Blacklock N,Matos-Ferreira A, Abranches-Monteiro L, Correia R, Lloyd S, Griffiths K.Lignans and isoflavonoids in plasma and prostatic fluid in men: Samplesfrom Portugal, Hong Kong, and the United Kingdom. Prostate 32: 122-128,1997

Non-patent Document 4: Tammy E H, Paul D M, Paul G F, Robert D, StephenB, Kenneth J, Ray M, Lorraine G O, Kristiina W, Holly M S, Karen J G.Long-term dietary habits affect soy isoflavone metabolism andaccumulation in prostatic fluid in caucasian men. J Nutr 135: 1400-1406,2005.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

The present inventors have found that fermented soybean hypocotylobtained by fermenting soybean hypocotyl with an equol-producingmicroorganism is usable as an equol-containing food material. Thefermented soybean hypocotyl contains not only equol but also isoflavone,saponin and like useful soybean-derived components. The fermentedsoybean hypocotyl achieves such effective physiological effectsattributable to these useful components, and therefore is usable as afunctional material. The present inventors also found that the fermentedsoybean hypocotyl is useful as an allergen-reduced material, because theallergens attributable to soybean hypocotyl therein are reduced. Thefermented soybean hypocotyl found by the present inventors exhibitsphysiological effects attributable to the useful components, and ishypoallergenic, and therefore it is usable as a functional foodmaterial.

In contrast, the content of the equol in the fermented soybean hypocotylvaries depending on the types of the soybean hypocotyl used for theproduction of the fermented soybean hypocotyl, types of theequol-producing microorganism, etc., but it is generally about 1 wt. %.If a material with an increased equol content can be provided, varioustypes of equol-containing foods become easily available in response tothe diversification of foods. However, the fermented soybean hypocotylitself is not a known product, and the effective method for efficientlyextracting useful component containing equol from fermented soybeanhypocotyl has not been found yet.

The fermented soybean hypocotyl, which is an equol-containing substanceobtained by the fermentation method describe above, is safer than thatobtained by a chemical synthesis method, and suitable for industrialproduction. However, equol-containing substances obtained by saidfermentation method contain metabolites other than equol and variouscomponents derived from raw materials remaining therein. Depending onthe type of raw material used for fermentation, the equol-containingsubstance obtained by a fermentation method may contain material thatcould become an allergen. Therefore, in order to use an equol as anadditive for foods or drugs, a technique not only for producing an equolbut also purifying the equol-containing substance so as to obtain ahighly pure equol is required. However, not many techniques for refiningequol have been reported yet, there is a demand for establishing atechnique, which is industrially applicable and by which an equol can bepurified easily and efficiently.

The fermented soybean hypocotyl has bitterness attributable to soybeanhypocotyl or equol. Therefore, sufficient attention must be paid to theflavor of the resulting food when the fermented soybean hypocotyl isused as a food material. The fermented soybean hypocotyl is a novel foodmaterial that has not been reported before, and it is unknown whichmethod is effective for improving the flavors thereof. In particular,when the fermented soybean hypocotyl is used as a material for bakedconfectioneries, it tends to suffer from a deterioration of flavor afterbeing subjected to a baking step. Therefore, exhibiting good flavor is avery difficult technical object.

An object of the present invention is to provide an extract, whichcontains useful equol-containing components and which is derived from anequol-containing fermented soybean hypocotyl, and a method for producingsuch an extract. The present invention provides a method for purifyingan equol-containing substance to effectively obtain a highly pure equol.Another object of the present invention is to provide a food materialobtained by fermenting an equol-containing fermented soybean hypocotylwith an equol-producing microorganism or its extract, in which theflavor is improved. Still another object of the present invention is toprovide a food product that contains an equol-containing fermentedsoybean hypocotyl and exhibits excellent flavor (in particular, bakedconfectioneries). Still another object of the present invention is toprovide various forms of foods that contain an equol-containingfermented soybean hypocotyl or its extract.

Means for Solving the Problem

The present inventors conducted intensive research to achieve the aboveobjects and found that by subjecting an equol-containing fermentedsoybean hypocotyl to extraction using an ethanol aqueous solution as anextractant, a useful equol-containing component can be efficientlyextracted from the fermented soybean hypocotyl. The present inventorsfurther found that by sequentially subjecting the equol-containingfermented soybean hypocotyl to extraction using an ethanol aqueoussolution and ethanol, the saponin content that causes an astringenttaste, bitterness, an acrid taste and like unpleasant tastes can bereduced, while effectively extracting equol and glycitein.

The present inventors also found that a highly pure equol can beobtained by suspending an equol-containing fermented soybean hypocotylin an aqueous solution having a pH of 2 to 6, collecting insolublematter, and then subjecting the resulting insoluble residue toextraction using an ethanol aqueous solution.

The present inventors further found that a highly pure equol can beefficiently and readily obtained by conducting the following Steps (1)to (5):

Step (1): subjecting an equol-containing substance to extraction usingan ethanol aqueous solution;

Step (2): subjecting the resulting extract to further extraction usingethanol;

Step (3): subjecting the resulting extract to extraction using hexane;

Step (4): subjecting the resulting insoluble residue to extraction usinga mixture of hexane and ether; and

Step (5): subjecting the resulting liquid extract to silica gel columnchromatography, and obtaining an equol-containing fraction.

The present inventors further found that a highly pure equol can beefficiently and readily obtained by conducting the following Steps (i)to (iii):

(i) subjecting the equol-containing substance to extraction using aspecific organic solvent or an aqueous organic solvent, andconcentrating the resulting liquid extract;

(ii) subjecting the resulting equol concentrate to silica gel columnchromatography, and obtaining an equol-containing fraction; and

(iii) removing the solvent from the resulting equol-containing fraction,and recrystallizing the resulting residue.

The present inventors also found that in a food material obtained bydispersing an equol-containing fermented soybean hypocotyl or itsextract in cacao mass, the bitterness attributable to theequol-containing fermented soybean hypocotyl can be reduced andexcellent flavor can be exhibited. The present inventors also found thatthe food material is usable in various foods, including bakedconfectioneries, without adversely affecting the flavor.

The present inventors successfully developed various food products intowhich an equol-containing fermented soybean hypocotyl or its extract isadded.

The present invention has been accomplished based on these findings andsome improvement added thereto.

The present invention provides a method for obtaining anequol-containing extract.

Item 1-1. A method for producing an equol-containing extract comprisingStep I-1:

subjecting an equol-containing fermented soybean hypocotyl to extractionusing an ethanol aqueous solution as an extractant; and collecting theresulting liquid extract.

Item 1-2. The method according to Item 1-1, which further comprises StepI-2:

removing the extractant from the liquid extract obtained in Step I-1;

conducting extraction using ethanol as an extractant; and collecting theliquid extract.

Item 1-3. The method according to Item 1-1, wherein the ethanol aqueoussolution used in Step I-1 has an ethanol concentration of 20 to 98 wt.%.

Item 1-4. The method according to any one of Items 1-1 to 1-3, whereinthe equol-containing fermented soybean hypocotyl is obtained byfermenting a soybean hypocotyl with an equol-producing microorganism.

Item 1-5. A method for producing an equol-containing extract fromequol-containing fermented soybean hypocotyl, comprising Steps I-1 andI-2;

Step II-1 of suspending the equol-containing fermented soybean hypocotylin water, adding an acid so as to adjust the pH to 2 to 6, andcollecting insoluble residue; and

Step II-2 of subjecting the insoluble residue obtained in Step II-1 toextraction using an ethanol aqueous solution as an extractant, and thencollecting the resulting liquid extract.

Item 1-6. The method of Item 1-5, wherein the acid used in Step II-1 isacetic acid.

The present invention provides the following equol-containing extracts.

Item 2-1. An equol-containing extract obtained by subjecting anequol-containing fermented soybean hypocotyl to extraction using anethanol aqueous solution as an extractant.

Item 2-2. An equol-containing extract obtained by sequentiallysubjecting an equol-containing fermented soybean hypocotyl to extractionusing an ethanol aqueous solution and ethanol.

Item 2-3. An equol-containing extract obtained by a method according toany one of Items 1-1 to 1-6.

The present invention provides the following methods for refining anequol.

Item 3-1. A method for purifying an equol-containing substance to obtainequol comprising Steps 1-1 to 1-5 below:

Step 1-1 of subjecting an equol-containing substance to extraction usingan ethanol aqueous solution as an extractant, and collecting theresulting liquid extract;

Step 1-2 of removing the solvent from the liquid extract obtained inStep 1-1, subjecting the resulting residue to extraction using ethanolas an extractant, and then collecting the liquid extract;

Step 1-3 of removing the solvent from the liquid extract obtained inStep 1-2, suspending the resulting residue in hexane, and thencollecting an insoluble residue;

Step 1-4 of subjecting the insoluble residue obtained in Step 1-3 toextraction using a mixture of hexane and ether as an extractant, andthen collecting the liquid extract; and

Step 1-5 of subjecting the liquid extract obtained in Step 1-4 to silicagel column chromatography, and obtaining an equol-containing fraction.

Item 3-2. The method according to Item 3-1, wherein the equol-containingsubstance is a fermented material that contains an equol.

Item 3-3. The method according to Item 3-2, wherein the fermentedmaterial that contains an equol is an equol-containing fermented soybeanhypocotyl material obtained by fermenting a soybean hypocotyl with anequol-producing microorganism.

Item 3-4. The method according to any one of Items 3-1 to 3-3, whereinthe extractant used in Step 1-4 is a mixture of hexane and diethylether.

Item 3-5. The method according to any one of Items 3-1 to 3-4, whereinthe extractant used in Step 1-4 is a mixture of hexane and ether havinga volume ratio of 10:90 to 20:80.

Item 3-6. The method according to any one of Items 3-1 to 3-5, whereinthe ethanol concentration of the ethanol aqueous solution used in Step1-1 is 20 to 98 vol. %.

Item 3-7. A method for purifying an equol-containing substancecomprising Steps 2-1 to 2-3 of:

Step 2-1 of subjecting the equol-containing substance to extractionusing, as an extractant, an organic solvent or an aqueous organicsolvent of at least one member selected from the group consisting ofethyl acetate, alcohol, acetone, dioxane, acetonitrile, diethyl ether,and toluene, and collecting the resulting liquid extract;

Step 2-2 of subjecting the liquid extract obtained in Step 2-1 to silicagel column chromatography, and obtaining an equol-containing fraction;and

Step 2-3 of removing the solvent from the equol-containing fractionobtained in Step 2-2, recrystallizing the resulting residue, andcollecting the deposited crystals.

Item 3-8. The method according to Item 3-7, wherein Step 2-3 comprises arecrystallization using an aqueous alcohol solution after therecrystallization using a mixture of ethyl acetate and hexane.

The present invention provides the following food materials, and foodsusing such food materials.

Item 4-1. A food material obtained by dispersing an equol-containingfermented soybean hypocotyl or its extract in cacao mass.

Item 4-2. The food material according to Item 4-1, wherein the foodmaterial contains 10 to 2000 parts by weight of cacao mass per 100 partsby weight of equol-containing fermented soybean hypocotyl or itsextract.

Item 4-3. The food material according to Item 4-1 or 4-2, wherein theequol-containing fermented soybean hypocotyl is obtained by fermentingsoybean hypocotyl with an equol-producing microorganism.

Item 4-4. The food material according to Item 4-3, wherein theequol-producing microorganism is a microorganism that can metabolize atleast one daidzein compound selected from the group consisting ofdaidzein glycoside, daidzein, and dihydrodaidzin to produce equol.

Item 4-5. The food material according to any one of Items 4-1 to 4-4,which takes a granular form, a chip form, or a plate-like form.

Item 4-6. An equol-containing food comprising a food material of any oneof Items 4-1 to 4-5.

Item 4-7. The equol-containing food according to Item 4-6, which is abaked confectionery.

The present invention provides the following foods:

Item 5-1. A food comprising an equol-containing fermented soybeanhypocotyl or its extract.

Item 5-2. A beverage comprising an equol-containing fermented soybeanhypocotyl or its extract.

Item 5-3. A dietary supplement comprising an equol-containing fermentedsoybean hypocotyl or its extract.

Item 5-4. A creamy food comprising an equol-containing fermented soybeanhypocotyl or its extract.

Item 5-5. A dessert comprising an equol-containing fermented soybeanhypocotyl or its extract.

Item 5-6. A snack comprising an equol-containing fermented soybeanhypocotyl or its extract.

Item 5-7. A seasoning comprising an equol-containing fermented soybeanhypocotyl or its extract.

Item 5-8. A retort-packed food comprising an equol-containing fermentedsoybean hypocotyl or its extract.

Item 5-9. A processed meat product comprising an equol-containingfermented soybean hypocotyl or its extract.

Item 5-10. A fish paste comprising an equol-containing fermented soybeanhypocotyl or its extract.

Item 5-11. A processed egg product comprising an equol-containingfermented soybean hypocotyl or its extract.

Item 5-12. A canned food comprising an equol-containing fermentedsoybean hypocotyl or its extract.

Item 5-13. A bread product comprising an equol-containing fermentedsoybean hypocotyl or its extract.

Item 5-14. A frozen dessert comprising an equol-containing fermentedsoybean hypocotyl or its extract.

Item 5-15. A processed soybean food comprising an equol-containingfermented soybean hypocotyl or its extract.

Item 5-16. A cooked rice product comprising an equol-containingfermented soybean hypocotyl or its extract.

Item 5-17. A soup comprising an equol-containing fermented soybeanhypocotyl or its extract.

Item 5-18. A food according to any one of Items 5-1 to 5-18, wherein theequol-containing fermented soybean hypocotyl is obtained by fermenting asoybean hypocotyl with an equol-producing microorganism.

Item 5-19. The food according to Item 5-18, wherein the microorganismhas an ability to metabolize at least one daidzein compound selectedfrom the group consisting of daidzein glycoside, daidzein, anddihydrodaidzin to produce equol.

Effects of the Invention

The method for producing an equol-containing extract of the presentinvention can produce an equol-containing extract that is useful as afunctional food material by effectively extracting useful componentscontaining equol from the equol-containing fermented soybean hypocotyl.By sequentially subjecting an equol-containing fermented soybeanhypocotyl to extraction using an ethanol aqueous solution and ethanol,an equol-containing extract that contains a high concentration of equoland glycitein, with a reduced amount of saponin that causes anunpleasant taste is obtainable. Accordingly, the equol-containingextract can be added to foods without adversely affecting the taste.

The purification method of the present invention makes it possible toreadily and efficiently obtain a highly pure equol from theequol-containing substance. In particular, the purification method ofthe present invention makes it possible to highly pure equol even whenthe equol-containing substance contains isoflavones whose structure issimilar to that of the equol by removing these isoflavones. Accordingly,the purification method of the present invention can be suitablyemployed in obtaining equol from an equol-containing fermented materialthat contains a large proportion of isoflavones.

Furthermore, the food material obtained by dispersing anequol-containing fermented soybean hypocotyl or its extract into cacaomass has reduced bitterness and exhibits excellent flavor, although itcontains an equol-containing fermented soybean hypocotyl or its extract.The food material of the present invention can be used in various foodswithout adversely affecting the flavor. When conventional food materialis added to a baked confectionery in a granular form or a chip form, thecomponents in the food material tend to diffuse into the entire bakedconfectionery in the baking step, and a desirable flavor may not beobtained. In the food material of the present invention, theequol-containing fermented soybean hypocotyl is covered with cacao mass.Therefore, even when the food material of the present invention is addedto a baked confectionery in a granular form or a chip form, diffusion ofthe effective components within the food material can be suppressed anda desirable flavor can be obtained.

The useful physiological benefits of the food of the present inventioncan be enjoyed based on the useful physiological activity of theequol-containing fermented soybean hypocotyl or its extract.

The equol-containing fermented soybean hypocotyl or its extract hasreduced soybean hypocotyl-derived allergens, and therefore the foodmaterial and various foods of the present invention can be safely takenby or administered to people suffering from soybean allergy.

BEST MODE FOR CARRYING OUT THE INVENTION

The following details the present invention.

1. Production Method for Equol-Containing Extract

The present invention provides a method for producing anequol-containing extract using an equol-containing fermented soybeanhypocotyl as a material. The production method for the equol-containingextract of the present invention is divided into the followingProduction Methods I and II. The following details an equol-containingfermented soybean hypocotyl used as a material of the method of thepresent invention, and describes in detail Production Methods I and II.

Equol-Containing Fermented Soybean Hypocotyl

In the production method for the equol-containing extract according tothe present invention, an equol-containing fermented soybean hypocotylis used as a raw material. The following describes an equol-containingfermented soybean hypocotyl.

An equol-containing fermented soybean hypocotyl is a kind of a fermentedsoybean hypocotyl produced by fermenting a soybean hypocotyl using anequol-producing microorganism.

The equol-producing microorganism used for the production of theequol-containing fermented soybean hypocotyl is selected frommicroorganisms having the ability (metabolic activity) to produce equolby metabolizing at least one daidzein compound selected from the groupconsisting of daidzein glycosides, daidzein, and dihydrodaidzin.Examples of the daidzein glycosides here include daidzein,malonyldaidzin, and acetyldaidzin.

The equol-producing microorganism is not limited and may be anymicroorganism having the foregoing ability. For example, any publiclyknown microorganisms, or any microorganisms obtained by a generalscreening method, may be used. It is known that such equol-producingmicroorganisms exist in the microorganisms belonging to Lactococcus,such as Lactococcus garvieae; microorganisms belonging to Streptococcus,such as Streptococcus intermedius or Streptococcus constellatus; ormicroorganisms belonging to Bacteroides such as Bacteroides ovatus.Among various kinds of equol-producing microorganism, the presentinvention prefers lactic acid bacteria belonging to Lactococcus orStreptococcus, further prefers lactic acid bacteria belonging toLactococcus, and in particular, lactic acid bacteria belonging toLactococcus garvieae. The equol-producing microorganisms may be isolatedfrom, for example, human feces, based on the index of the existence ofan equol-producing property. The inventors of the present inventiondeposited some identified bacterium that had been isolated from humanfeces; namely, Lactococcus 20-92 (FERM BP-10036), Streptococcus E-23-17(FERM BP-6436), Streptococcus A6G225 (FERM-6437), and BacteroidesE-23-15 (FERM BP-6435), that are available to be used as theequol-producing microorganisms. Among these, the present inventionparticularly prefers Lactococcus 20-92.

The equol-containing fermented soybean hypocotyl is produced usingsoybean hypocotyl as a fermenting material. The soybean hypocotyldesignates a part corresponding to the plumule or the radicle on thegermination of soybean, and is known to contain a large amount ofdaidzein compounds such as daidzein glycosides or daidzein. The soybeanhypocotyl used in the present invention is not limited by the producingdistrict of soybean or whether processed or unprocessed, unless thedaidzein compounds inside are not significantly lost. For example, theequol-containing fermented soybean hypocotyl may be a raw hypocotyl, ormay be one isolated from heated, dried or steam-boiled soybean, or oneobtained by heating, drying or steam-boiling hypocotyl that is isolatedfrom unprocessed soybean. Further, the soybean hypocotyl may beprocessed by degreasing or deproteinization. The form of the soybeanhypocotyl is not particularly limited, and may be in the form of powder,chunks, or pulverized or fragmentized grains. Powdery soybean hypocotylis particularly preferable because of its efficient equol production.The fermentation of soybean hypocotyl is carried out by first adjustingthe water content of the soybean hypocotyl by adding an appropriateamount of water, and then inoculating the equol-producing microorganismto the hypocotyl.

The amount of water added to the soybean hypocotyl is adjusted dependingon the type of equol-producing microorganism or the type of fermenter.The ratio of water to the soybean hypocotyl in the beginning offermentation is 400 to 4,000 parts by weight, preferably 500 to 2,000parts by weight, further preferably 600 to 1,000 parts by weight, basedon 100 parts by weight (dry weight) of the soybean hypocotyl.

Further, if necessary, some additives may be added to the soybeanhypocotyl as the raw material in the fermentation to improve thefermentation efficiency or flavor of the product. Examples of theadditives include nitrogen sources such as a yeast extract, polypeptone,or a meat extract; carbon sources such as glucose or sucrose; inorganicsalts such as phosphate, carbonate or hydrosulfate; vitamins; andnutritional components such as amino acid. Particularly, when using anequol-producing microorganism for converting arginine into ornithine(hereinafter referred to as an “ornithine/equol-producingmicroorganism”), arginine is added to the soybean hypocotyl beforefermentation so that the fermented substance contains ornithine. In thiscase, the amount of arginine is, for example, about 0.5 to 3 parts byweight, based on 100 parts by weight (dry weight) of the soybeanhypocotyl. The ornithine/equol-producing microorganism may be obtainedby a publicly-known screening method using an index of theequol-producing ability and the conversion ability from arginine intoornithine. The ornithine/equol-producing microorganism may be selectedfrom the group of Lactococcus garvieae, typified by Lactococcus 20-92(FERM BP-10036).

Further, the pH of the fermenting material (a soybean hypocotylcontaining substance) is not particularly limited within a range for theequol-producing microorganism to grow; however, to secure excellentproliferation of the equol-producing microorganism, the pH of thefermenting material preferably falls to within about 6 to 7, morepreferably about 6.3 to 6.8.

Further, isoflavone containing the above-mentioned daidzein compoundsmay be added to the fermenting material (a soybean hypocotyl containingsubstance). Such inclusion of isoflavone in the fermenting materialincreases the equol content of the fermented soybean hypocotyl, therebyimproving the usability of the fermented soybean hypocotyl.

The fermentation of the soybean hypocotyl is carried out underappropriate conditions according to the growing characteristic of theequol-producing microorganisms. For example, when the equol-producingmicroorganisms listed above are used, the fermentation of the soybeanhypocotyl is carried out under anaerobic conditions.

The fermentation temperature is not limited within an appropriate rangefor the equol-producing microorganism to grow. The temperature isgenerally 20 to 40° C., preferably 35 to 40° C., and more preferably 36to 38° C.

The fermentation time is determined depending on the desired productionamount of equol, the residual amount of daidzein compounds, the type ofthe equol-producing microorganism or the like. The fermentation time isgenerally 1 to 10 days, preferably 2 to 7 days, and further preferably 3to 5 days.

In the fermented soybean hypocotyl obtained through the fermentationunder the foregoing conditions, the produced equol is accumulated, andthereby the useful physiologic activity of equol can be expressed. Theequol content in the fermented soybean hypocotyl varies depending on thetype of the equol-producing microorganism or fermentation conditions.The equol content in the fermented soybean hypocotyl is generally 1 to20 mg, preferably 2 to 12 mg, and more preferably 5 to 8 mg, per gram(dry weight) of the fermented soybean hypocotyl.

Apart from equol, the equol-containing fermented soybean hypocotyl alsocontains various isoflavones such as daidzein compounds includingdaidzin, malonyldaidzin, acetyldaidzin, daidzein, or dihydrodaidzin(these components are referred to as “daidzein compounds”, hereinafter);genistein compounds including malonylgenistin, acetylgenistin,genistein, dihydrogenistin (these components are referred to as“genistein compounds”, hereinafter); or glycitein compounds includingglycitin, malonylglycitin, acetylglycitin, glycitein, dihydroglycitein(these components are referred to as “glycitein compounds” hereafter).The isoflavones also express the useful physiologic activities. Thecontent of each isoflavone (incl. equol) in the fermented soybeanhypocotyl is generally 5 to 20 mg, preferably 5 to 15 mg, and morepreferably 8 to 15 mg, per gram (dry weight) of the equol-containingfermented soybean hypocotyl.

Further, the equol-containing fermented soybean hypocotyl also differsfrom general soybean hypocotyls in terms of the composition ofisoflavones other than equol. Particularly, the total content ofgenistein compounds in the equol-containing fermented soybean hypocotylis preferably not more than 15 wt. %, and more preferably not more than12 wt. %. With such a low genistein compound content, theequol-containing fermented soybean hypocotyl is more advantageous thanthe unfermented soybean hypocotyl in terms of the composition ofisoflavones. Note that genistein compounds are kinds of isoflavone andare chemicals suspected of endocrine disruption, and there is a demandfor the reduction in genistein compounds concentrations in food (Young HJ, Jodi F, Kimberly F A, Daniel R D, William G H.

Effects of dietary daidzein and its metabolite, equol, at physiologicalconcentrations on the growth of estrogen-dependent human breast cancer(MCF-7) tumors implanted in ovariectomized athymic mice. SeeCarcinogenesis 127(4):856-863, 2006). The following is an isoflavonecomposition example of the equol-containing fermented soybean hypocotyl(the unit “mg” represents the total amount of each isoflavone per gram(dry weight) of the fermented soybean hypocotyl).

Equol: 1 to 20 mg, preferably 2 to 12 mg

Daidzein compounds: 0.1 to 30 mg, preferably 0.1 to 1.5 m

Genistein compounds: 0.05 to 2.5 mg, preferably 0.05 to 2 mg

Glycitein compounds: 0.1 to 4 mg, preferably 2 to 3.5 mg.

The following is a composition ratio example of the isoflavones in theequol-containing fermented soybean hypocotyl (the unit “wt. %”represents a ratio relative to the total amount of isoflavones in theequol-containing fermented soybean hypocotyl).

Equol: 30 to 75 wt. %, preferably 40 to 70 wt. %, more preferably 45 to70 wt. %

Daidzein compounds: 1 to 20 wt. %, preferably 2 to 15 wt. %, morepreferably 4 to 12 wt. %

Genistein compounds: 0.1 to 20 wt. %, preferably 1 to 15 wt. %, morepreferably 1 to 10 wt. %

Glycitein compounds: 10 to 50 wt. %, preferably 15 to 35 wt. %, morepreferably 25 to 35 wt. %.

Since this equol-containing fermented soybean hypocotyl has a particularisoflavone composition that is not found in any prior art, it can bedefined as an isoflavone-containing substance specified by the foregoingisoflavone composition.

Lactococcus 20-92 (FERM BP-10036) is particularly preferred as anequol-producing microorganism in the production of the equol-containingfermented soybean hypocotyl having the foregoing isoflavone composition.

The equol-containing fermented soybean hypocotyl also contains saponinderived from the soybean hypocotyl. The equol-containing fermentedsoybean hypocotyl generally contains saponin in an amount of 10 to 80mg, preferably 20 to 50 mg, and more preferably 30 to 40 mg, per gram(in dry weight) of the equol-containing fermented soybean hypocotyl.

Further, as described above, ornithine is contained in the fermentedsoybean hypocotyl produced from fermentation with anornithine/equol-producing microorganism after adding arginine to soybeanhypocotyl. The ornithine content of the equol-containing fermentedsoybean hypocotyl is 5 to 20 mg, preferably 8 to 15 mg, and morepreferably 9 to 12 mg, per gram (dry weight) of the equol-containingfermented soybean hypocotyl.

The equol-containing fermented soybean hypocotyl obtained throughfermentation under the foregoing conditions may be used directly afterfermentation as a material of the production method of the presentinvention, or may be dried after fermentation to be a dry solid beforebeing used as the material of the production method of the presentinvention.

Further, the equol-containing fermented soybean hypocotyl obtainedthrough fermentation under the foregoing conditions may be subjected topreprocessing, such as defatting, before the following Step I-1. Thedefatting can be performed by a method using hexane as an extractant.

1-1. Production Method I

In Production Method I, the production of the equol-containing extractis accomplished by carrying out Step I-1, which is described below.

Step I-1

Production Method I for producing the equol-containing extract comprisesa Step I-1 of extracting an equol-containing fermented soybean hypocotylusing an aqueous ethanol solution as an extractant, and collectingliquid extract.

The ethanol concentration of the aqueous ethanol solution (a mixedsolution of ethanol and water) used in Step I-1 is generally 20 to 98vol. %, preferably 45 to 95 vol. %, and more preferably 65 to 85 vol. %.By meeting this concentration range, it becomes possible to extractuseful components including equol from the equol-containing fermentedsoybean hypocotyl.

The extraction in Step I-1 uses an aqueous ethanol solution in an amountof, for example, 1 to 1000 parts by weight, preferably 2 to 500 parts byweight, more preferably 5 to 20 parts by weight, per part by weight (dryweight) of the equol-containing fermented soybean hypocotyl.

The Step I-1 extraction conditions are not particularly limited, and awarm or cold dipping extraction method, percolation method or the likemay be used. More specifically, an aqueous ethanol solution is added tothe equol-containing fermented soybean hypocotyl, and the mixture isallowed to stand or stirred for at least one minute at room temperature,preferably for 5 minutes to 48 hours at room temperature.

After such extraction, the liquid extract is isolated to obtain anequol-containing extract. The collection of the liquid extract isperformed by a publicly-known method, such as filtration orcentrifugation.

The liquid extract thus isolated in Step I-1 contains useful componentsincluding equol and glyciteins, and the amount of oil is less than thatin the equol-containing fermented soybean hypocotyl. The liquid extractthus obtained in Step I-1 may be directly incorporated into a food ormedicine as a liquid equol-containing extract, or may be solidified byremoving the extractant before incorporated into a food or medicine.Further, the liquid extract thus obtained in Step I-1, either in theform of a liquid or a solid equol-containing extract, may be subjectedto equol refining.

The liquid extract isolated in Step I-1 contains equol, glyciteins, andsaponin, which can cause unpleasant astringency, bitterness, oracridness. It is thus preferable to subject the liquid extract isolatedin Step I-1 to the following Step I-2 so as to remove saponin andincrease the concentration of the useful components, including equol.

Step I-2

Step I-2 comprises removing the extractant from the liquid extractobtained in Step I-1, carrying out another extraction using ethanol asan extractant, and collecting an extract.

In Step I-2, the removal of the extractant from the extract obtained inStep I-1 can be performed by a publicly-known method such as drying.

The solid obtained by the removal of the extractant from the extractobtained in Step I-1 (this solid may be hereinafter referred to as“Solid Material”) is subjected to extraction using ethanol as anextractant. The ethanol used as an extractant in Step I-2 may bewater-free ethanol that however may inevitably contain vapor.

The extraction in Step I-2 uses, for example, 1-1000 parts by weight,preferably 5-500 parts by weight, and more preferably 5-20 parts byweight of ethanol, per part by weight (dry weight) of the solidmaterial.

The condition in Step I-2 extraction is not particularly limited, andmay be performed by a warm or cold dipping extraction method,percolation method or the like. More specifically, ethanol is added tothe solid material and the mixture is allowed to stand or stirred for atleast one minute at room temperature, preferably for 5 minutes to 48hours at room temperature. The Step I-2 extraction may be combined withsupersonic treatment to increase the equol collection rate.

After such extraction, the extract is isolated to obtain anequol-containing extract. The collection of the extract is performed bya publicly-known method, such as filtration or centrifugation.

The extract thus isolated in Step I-2 contains a greater amount ofuseful components including equol and glyciteins, and a reduced amountof saponin. The usefulness of this extract is therefore greater than theextract obtained in Step I-1. The extract thus obtained in Step I-2 maybe directly incorporated into a food or medicine as a liquidequol-containing extract, or may be solidified by removing theextractant before being incorporated into a food or medicine. Theextract thus obtained in Step I-2, either in the form of a liquid or asolid equol-containing extract, may be subjected to equol refining. Notethat, in order to improve the conservation stability of theequol-containing extract, it is more preferable to solidify the liquidextract by removing the extractant.

Composition and Use of Equol-Containing Extract Obtained by ProductionMethod I

The equol content of the liquid extract obtained in Step I-1 or Step I-2is 0.2 to 40 wt. %, preferably 0.4 to 24 wt. %, and more preferably 1 to16 wt. %, based on the total amount of a dry solid product (100 wt. %).The glycitein content of the liquid extract obtained in Step I-1 or StepI-2 is 0.01 to 8 wt. %, preferably 0.08 to 5 wt. %, and more preferably0.2 to 3 wt. %, based on the total amount of a dry solid product.

The equol-containing extract obtained in Step I-1 or Step I-2 containsequol at a high concentration, and expresses the useful activities basedon equol. More specifically, the equol-containing extract obtained inStep I-1 or Step I-2 has an effect of preventing or treating variousdiseases or symptoms including menopausal disorders, osteoporosis,prostatic hypertrophy, and metabolic syndrome; or for blood cholesterollevel reduction, skin-whitening, pimples, intestinal control, obesity,and diuresis. Since the useful activities are thus expressed, theequol-containing extract obtained in Step I-1 or Step I-2 may beincorporated into food or medicines.

When used for a foodstuff, the equol-containing extract obtained in StepI-1 or Step I-2 is often incorporated into beverages, granules, finegranules, capsules, tablets, powder, dairy products, gums, gummycandies, puddings, bars, and other various dry foods. The foodcontaining the equol-containing extract may be used as a general foodproduct, and also as a specified health food, nutritional supplement,functional food, invalid food or the like. Particularly, any foodcontaining the fermented soybean hypocotyl of the present invention isuseful as a nutritional supplement. Note that the details of foodstuffscontaining the equol-containing extracts obtained in Step I-1 or StepI-2 are described later.

When the equol-containing extract obtained in Step I-1 or Step I-2 isincorporated into a foodstuff, the proportion of the equol-containingextract in the food varies depending on the type of food, the equolcontent, the age and sex of the consumer, the desired effect etc.Typically, the total amount (dry weight base) of the equol-containingextract is 0.1 to 90 g, per 100 g of food.

When used for a medicinal product, the equol-containing extract obtainedin Step I-1 or Step I-2 is often incorporated into tablets, pills,powder, liquids, suspensions, emulsions, granules, capsules, orsuppositories. Such a medicinal product containing the equol-containingextract has an effect of preventing or treating various diseases orsymptoms including menopausal disorders (indefinite menopausalcomplaints, osteoporosis, hyperlipidemia), osteoporosis, prostatichypertrophy, and metabolic syndrome; or for blood cholesterol levelreduction, skin-whitening, pimples, intestinal control, obesity, anddiuresis. Particularly, the medicinal product containing theequol-containing extract is suitable for the prevention or treatment ofindefinite complaint or post-menopausal symptoms (e.g. osteoporosis,menopausal disorder) of middle-aged women.

The dose of the medicinal product containing the equol-containingextract obtained in Step I-1 or Step I-2 cannot be uniformly defined, asit varies depending on the equol content in the equol-containingextract, age, weight, symptom of the object or the dosage number. Theadult dosage per day typically corresponds to the amount (dry weight) ofthe equol-containing extract from 1 to 2000 mg, preferably 5 to 1000 mg,and more preferably 40 to 50 mg. It is particularly preferable todetermine the dose of the equol-containing extract so that 2 to 30 mg ofequol is administered per day.

1-2. Production Method II

Production Method II produces an equol-containing liquid extract bycarrying out the following Step II-1 and Step II-2. The followingdetails Production Method II.

Step II-1

Production Method II first suspends an equol-containing fermentedsoybean hypocotyl in water, adds an acid to the suspension to adjust thepH to a range of 2 to 6, and isolates an insoluble residue (Step II-1).In other words, in Step II-1, an equol-containing fermented soybeanhypocotyl is suspended in an aqueous solution (pH 2 to 6) containing anacid, thereafter collecting an insoluble residue.

In Step II-1, an equol-containing fermented soybean hypocotyl issuspended in water, and an acid is added to the suspension to adjust thepH to a range of from 2 to 6.

The acid used in Step II-1 may be an organic acid or an inorganic acid.The organic acid is not limited as long as it is adjustable to theforegoing pH range. Examples of the organic acid include citric acids,L-tartaric acids, fumaric acids, L-ascorbic acids, gluconic acids,acetic acids, lactic acids, DL-malic acids, oxalic acids, formic acids,propionic acids, butanoic acids, and methanesulfone acids. Similarly,the inorganic acid is not limited as long as it is adjustable to theforegoing pH range. Examples of the inorganic acid include hydrochloricacids, sulfuric acids, nitric acids and phosphoric acids. Among them,acetic acids, hydrochloric acids and sulfuric acids are preferred.

In Step II-1, the amount of water in which the equol-containingfermented soybean hypocotyl is suspended is, for example, 3 to 30 partsby weight, more preferably 5 to 20 parts by weight, per part by weight(dry weight) of an equol-containing fermented soybean hypocotyl.

In Step II-1, an equol-containing fermented soybean hypocotyl is mixedin an aqueous solution 2 to 6 in pH, and the solution is stirred orallowed to stand at room temperature for at least 30 minutes, morepreferably 2 to 24 hours.

Such treatment leaves equol as a part of the insoluble residue, and theequol can be obtained by collecting the insoluble residue using apublicly-known method such as filtration or centrifugation.

The insoluble residue thus obtained in Step II-1 is then subjected tothe following Step II-2.

Step II-2

The insoluble residue thus obtained in Step II-1 is subjected to anotherextraction using an aqueous ethanol solution as an extractant. Theresulting liquid extract is isolated (Step II-2).

The insoluble residue obtained in Step II-1 may be subjected to StepII-2, regardless of whether it is a dry product or a wet product.However, when the insoluble residue is a wet product, it is preferableto adjust the water content of the aqueous ethanol solution (describedlater) in consideration of the measured water content of the insolublematter.

The ethanol concentration of the aqueous ethanol solution (a mixedsolution of ethanol and water) used in Step II-2 is, for example, 50 to95 vol. %, more preferably 70 to 90 vol. %. With this concentrationrange, the equol extraction obtained from the insoluble residue in StepII-2 can be efficiently performed.

The extraction in Step II-2 uses an aqueous ethanol solution in anamount of, for example, 2 to 30 parts by weight, more preferably 4 to 10parts by weight, per part by weight (dry weight) of the insolubleresidue obtained in Step II-1.

The condition in the extraction in Step II-2 is not particularlylimited, and the warm or cold dipping extraction method, percolationmethod or the like may be used. More specifically, an aqueous ethanolsolution is added to the insoluble residue obtained in Step II-1, andthe mixture is allowed to stand or stirred for at least 30 minutes atroom temperature, preferably 1 minute to 50 hours at room temperature.

After such extraction, the liquid extract is isolated to obtain anequol-containing fraction. The collection of the extract is performed bya publicly-known method such as filtration or centrifugation.

Because of the inclusion of concentrated equol, the liquid extract thusisolated in the Step II-2 contains equol at a high concentration anduseful components such as glysitein compounds, the extract is moreuseful than the equol-containing fermented soybean hypocotyl. The liquidextract obtained in Step II-2 may be directly incorporated into a foodor medicine as a liquid equol-containing extract, or may be processedinto powder by removing the extractant before incorporated into a foodor medicine. The liquid extract thus obtained in Step II-2, either inthe form of a liquid or a solid equol-containing extract, may besubjected to equol refining. Note that, in order to improve theconservation stability of the equol-containing extract, it is morepreferable to solidify the liquid extract by removing the extractant.

The removal of the extractant from the liquid extract obtained in StepII-2 may be performed under the same conditions as in Step I-2 ofProduction Method 1; however, the removal is preferably performed byspray-drying.

Composition and Use of Equol-Containing Extract Obtained by ProductionMethod II

By drying the liquid extract obtained in Method II (the liquid extractobtained in the foregoing Step II-2), it is possible to isolate equolwith a collection rate of 90% or greater. Thus obtained isequol-containing powder that contains equol ten times greater than theequol in an equol-containing fermented soybean hypocotyl. With this highequol content, the equol-containing powder more effectively expressesthe useful activities derived from equol. The purpose or method of useof the equol-containing powder obtained in Method II is the same as thatof the equol-containing powder obtained in Method I.

2. Equol Purificaton Method

The present invention also provides a method for purifying anequol-containing substance to obtain equol. The equol purificationmethod of the present invention is divided into the followingPurification Methods 1 and 2. The following details an equol-containingfermented soybean hypocotyl to be refined by the purification method ofthe present invention and describes in detail Purification Methods 1 and2.

Equol-Containing Substance

In the purification method of the present invention, theequol-containing substance to be subjected to the purification processis not limited as long as it contains equol. For example, theequol-containing substance used for the method may be a reaction productcontaining equol generated by chemical synthesis, or an equol-containingfermented substance produced by fermentation. The equol-containingfermented substance obtained through fermentation contains many kinds ofisoflavone similar in structure to equol. However, since the method ofthe present invention can advantageously separate equol from isoflavonein a efficient manner, an equol-containing fermented substance issuitable for an equol-containing substance for the purification methodof the present invention.

The following explains an equol-containing fermented substance.

The equol-containing fermented substance was produced through apublicly-known fermentation method using an equol-producingmicroorganism. More specifically, a microorganism with the ability(metabolic activity) to produce equol by metabolizing at least onedaidzein compound selected from the group consisting of daidzeinglycosides, daidzein, and dihydrodaidzin, is inoculated in a fermentingmaterial (material to be subjected to the fermentation) containing thedaidzein. The sample is then fermented (cultured) under the conditionssuitable to grow the microorganism. The resulting fermented substancecontains equol.

The equol-producing microorganism is selected from the list of“Equol-Containing Fermented Soybean Hypocotyl” in the section “1.Production Method for Equol-Containing Extract”.

The fermenting material containing daidzein compounds is not limited byother factors; however, the material is preferably approved for itssafety as a food material. Examples of the fermenting materialcontaining daidzein compounds include soybeans, soybean hypocotyl,soybean hypocotyl extract, tofu, deep-fried tofu, soy milk, fermentedsoybeans, soy sauce, bean paste, a tempeh, and a red clove or itsextract, alfalfa or its extract. A suitable fermenting materialcontaining daidzein compounds is a soybean hypocotyl because of its highdaidzein content.

Further, an isoflavone containing any of above-described daidzeincompound may be added in advance to the fermenting material containingdaidzein compounds. With this extra addition of isoflavone to thefermenting material, the equol content of the resulting fermentedsubstance increases.

Further, as required, some additives may be used in the fermentingmaterial containing daidzein compounds to improve fermentationefficiency etc. Examples of the additives include nitrogen sources suchas a yeast extract, poly peptone, or a meat extract; carbon sources suchas glucose or sucrose; inorganic salts such as phosphate, carbonate orhydrosulfate; vitamins; and nutritional components such as amino acid.

Various fermentation conditions including water content, time,temperature, atmosphere in the production of an equol-containingfermented substance are determined depending on the type of theequol-producing microorganism, the type of the fermenting material, theproduction amount of equol, the residual daidzein compound amount, orthe like.

For the equol-containing substance used for the purification method ofthe present invention, the equol-containing fermented soybean hypocotyldescribed in the section “1. Production Method for Equol-ContainingExtract” is particularly preferable.

2-1. Purification Method 1

The Purification Method 1 refines equol by carrying out the followingSteps 1-1 to 1-5.

Step 1-1

The purification method of the present invention carries out extractionwith respect to an equol-containing substance using an aqueous ethanolsolution as an extractant, and then collects the resulting liquidextract (Step 1-1).

A typical ethanol concentration of the aqueous ethanol solution used inStep 1-1 is 20 to 98 vol. %, preferably 45 to 95 vol. %, and morepreferably 45 to 60 vol. %. Ensuring this ethanol concentration, it ispossible to efficiently extract equol from the equol-containingsubstance.

The extraction in Step 1-1 uses an aqueous ethanol solution in an amountof, for example, 1 to 1000 parts by weight, preferably 2 to 500 parts byweight, more preferably 5 to 20 parts by weight, per part by weight (dryweight) of the equol-containing substance.

The conditions in the extraction in Step 1-1 are not particularlylimited, and a warm or cold dipping extraction method, percolationmethod or the like may be used. More specifically, an aqueous ethanolsolution is added to the equol-containing substance, and the mixture isallowed to stand or stirred for at least one minute at room temperature,more preferably 5 minutes to 48 hours at room temperature.

After such extraction, the liquid extract is isolated to obtain anequol-containing fraction. The collection of the liquid extract isperformed by a publicly-known method such as filtration orcentrifugation.

The liquid extract thus isolated in Step 1-1 contains a reduced amountof oil in the equol-containing substance, and isoflavone includingglycitein compounds and saponin, as well as equol.

The liquid extract thus obtained in Step 1-1 is subjected to thefollowing Step 1-2.

Step 1-2

After removing the extractant from the liquid extract obtained in Step1-1, the residue is subjected to extraction using ethanol as anextractant, and the liquid extract is isolated (Step 1-2).

In Step 1-2, the removal of the extractant from the liquid extractobtained in Step 1-1, and another extraction of the thus-obtainedresidue using an ethanol, and the collection of the liquid extract arecarried out in the same conditions as those of Step I-2 of ProductionMethod I in the section “1. Production Method for Equol-ContainingExtract”.

The liquid extract collected in Step 1-2 contains a reduced amount ofsaponin, and contains isoflavone including glycitein compounds, as wellas equol. The liquid extract thus obtained in Step 1-2 is subjected tothe following Step 1-3.

Step 1-3

After removing the extractant from the liquid extract obtained in Step1-2, the residue is suspended in hexane to isolate an insoluble matter(Step 1-3).

In Step 1-3, the removal of extractant from the liquid extract obtainedin Step 1-2 may be performed by a publicly-known method such asdistillation under reduced pressure, drying or the like.

The extractant is removed from the liquid extract obtained in Step 1-2,and hexane is added to the residue (hereinafter occasionally referred toas “second material residue”) to suspend the residue in hexane.

The extraction in Step 1-3 uses hexane in an amount of 1 to 50 parts byweight, and more preferably 5-10 parts by weight, per part by weight(dry weight) of the second material residue.

The conditions for the extraction in Step 1-3 are not particularlylimited. For example, a warm or cold dipping extraction method may beadopted. More specifically, hexane is added to the second materialresidue, and the mixture is allowed to stand or stirred for at least 5minute at room temperature, preferably 3 minutes to an hour at roomtemperature.

After such suspension using hexane, equol is extracted as an insolublematter. Therefore, by collecting the insoluble matter, anequol-containing extract is obtained. The collection of the insolubleresidue is performed by a publicly-known method such as filtration orcentrifugation.

Through Step 1-3, lipid-soluble impurities are removed from the liquidextract obtained in Step 1-2. The insoluble residue obtained in Step 1-3is subjected to the following Step 1-4.

Step 1-4

Step 1-4 comprises subjecting the insoluble residues obtained in Step1-3 to extraction using a mixed solution of hexane and ether as anextractant, and collecting the resulting liquid extract (Step 1-4).

The ether, as one of the components of the extractant used in Step 1-4,is not particularly specified. Examples of the ether includedimethylether, methylethylether, and diethylether, and they may be usedsingly or in combination. Among them, diethylether is particularlypreferred because of its equol refining efficiency.

The mixture ratio of the mixed solution of hexane and ether used in Step1-4 is not particularly limited; however, to obtain equol of desiredpurity, the ratio of hexane to ether in volume is 10:90 to 20:80, andpreferably 30:70 to 25:75.

The extraction in Step 1-4 uses the mixed solution of hexane and etherin an amount of 50 to 5000 parts by weight, more preferably 200 to 1000parts by weight, per part by weight (dry weight) of the insolubleresidue obtained in Step 1-3.

The conditions for the extraction in Step 1-4 are not particularlylimited. For example, a warm or cold dipping extraction method orpercolation method may be adopted. More specifically, the mixed solutionof hexane and ether is added to the insoluble matter obtained in Step1-3, and the mixture is allowed to stand or stirred for at least 10minutes at room temperature, preferably 15 minutes to 30 hours at roomtemperature.

After such extraction, the liquid extract is isolated to obtain equol.The collection of the liquid extract is performed by a publicly-knownmethod such as filtration or centrifugation.

Step 1-5

The liquid extract obtained in Step 1-4 is subjected to silica gelchromatography to obtain an equol-containing fraction (Step 1-5).

Step 1-5 may adopt any kind of silica gel chromatography that usesseparate columns each containing silica gel as a filler. Examples ofsilica gel column chromatography include open column chromatography thatcauses a solvent flow by gravity drop, flash column chromatography thatmakes a solvent flow using a pump, and high-performance liquidchromatography. Open column chromatography and flash columnchromatography are particularly preferred, as they can be easily carriedout in an industrial manner. When carrying out silica gel columnchromatography, it is preferable to use a mixed solution of hexane andether as a mobile phase (developing solvent) to further improve theequol refining degree. The ether used for the mobile phase is notlimited. Examples of the ether include dimethylether, methylether, anddiethylether, and they may be used solely or in combination. To improvethe equol refining degree, the ether is preferably the same as that usedfor the mixed solution of hexane and ether in Step 1-4. In the elutionof equol using silica gel column chromatography in Step 1-5, it ispreferable to use a mixed solution of hexane and ether identical incomposition to that used in Step 1-4 as the initial mobile phase, andthe ratio of ether is gradually increased according to the gradientmethod. Such an increase of the ratio of ether in the mobile phasecauses the equol to separate from other impurities, together with themobile phase in which hexane and ether is mixed at 40:60 to 20:80, morepreferably 30:70 to 25:50. Thereafter, the fraction containing equolhaving been eluted by silica gel column chromatography is isolated toobtain highly pure equol. Then, the extractant is removed from theequol-containing fraction by a publicly-known method such asdistillation under reduced pressure, drying or the like. This removesmost of the impurities from the fraction, thereby leaving a highly pureequol solid.

2-2. Prification Method 2

Step 2-1

Purification method 2 of the present invention carries out extractionwith respect to an equol-containing substance using, as an extractant,an organic solvent or an aqueous organic solvent of at least one memberselected from the group consisting of ethyl acetate, alcohol, acetone,dioxane, acetonitrile, diethylether, and toluene, and then concentratesthe liquid extract (Step 2-1).

In Step 2-1, the extractant may be an organic solvent of at least onemember selected from the group consisting of ethyl acetate, alcohol,acetone, dioxane, acetonitrile, diethylether, and toluene. However, toensure efficient equol extraction, it is more preferable to use anaqueous organic solvent that is obtained by adding water to theaforementioned organic solvent.

Examples of the alcohol used as an extractant include methanol, ethanol,isopropyl alcohol, and n-propyl alcohol.

A typical concentration of the aqueous organic solvent is 80 to 99 vol.%, preferably 90 to 97 vol. %. Ensuring this concentration, it ispossible to obtain a liquid extract with a high equol content.

One preferable extractant used in Step 2-1 is aqueous ethyl acetate.

The extraction conditions are not particularly limited, and a warm orcold dipping extraction method or the like may be used. Morespecifically, an extractant is added to the equol-containing substance,and the mixture is allowed to stand or stirred for at least 30 minutesat room temperature, more preferably for 1 hour to 50 hours at roomtemperature.

The conditions in the Step 1-1 extraction are also not particularlylimited, and a warm or cold dipping extraction method, percolationmethod or the like may be used.

After such extraction, equol educed in the liquid extract is isolated bya publicly-known solid-liquid dividing method such as filtration orcentrifugation. The obtained liquid extract is then concentrated, forexample, under reduced pressure to obtain an equol concentrate. Theequol concentrate thus obtained is subjected to the following Step 2-2.

Step 2-2

The equol concentrate obtained in Step 2-1 is subjected to silica gelcolumn chromatography to obtain an equol-containing fraction (Step 2-2).

In Step 2-2, the equol concentrate obtained in Step 2-1 may be directlysubjected to silica gel column chromatography.

Further, if the extraction in Step 2-1 is carried out with an aqueoussolvent containing alcohol, an equivalent amount of ethyl acetate andwater that is 5 to 10 times in volume the concentrated liquid extractare added to the concentrate, and the ethyl acetate fraction isseparated and concentrated to be subjected to column chromatography.This column chromatography in Step 2-2 may be any kind of chromatographythat uses separate columns each containing silica gel as a filler.Examples of silica gel column chromatography include open columnchromatography that causes a solvent flow by gravity drop, flash columnchromatography that makes a solvent flow using a pump, andhigh-performance liquid chromatography. Open column chromatography andflash column chromatography are particularly preferred, as they can beeasily carried out in an industrial manner.

In the silica gel column chromatography in Step 2-2, it is preferable touse a mixed solution of an ethyl ether or ethyl acetate solution(Solution A, hereinafter) and an n-hexane, petroleum ether or n-heptanesolution (Solution B, hereinafter) as a mobile phase. The ratio ofSolution B in the mixed solution is 2 to 50 parts by volume, morepreferably 3 to 10 parts by volume, per part by volume of Solution A.

The liquid extract obtained in Step 2-1 is thus subjected to silica gelcolumn chromatography to isolate the equol-containing fraction.

The equol-containing fraction obtained in Step 2-2 is subjected to Step2-3

Step 2-3

The solvent is removed from the equol-containing fraction obtained inStep 2-2, and the resulting residue is subjected to recrystallization.The precipitated crystals are then isolated (Step 2-3).

The removal of a solvent from the equol-containing fraction obtained inStep 2-2 may be performed by a publicly-known solvent removal method,such as concentration under reduced pressure.

The solvent used for the recrystallization in Step 2-3 is notparticularly limited as long as it can be used for the recrystallizationof equol. A typical solvent is a mixed solution of ethanol and water(aqueous ethanol solution), ethyl acetate, a mixed solution of ethylacetate and hexane, or a mixed solution of ethanol and hexane. The mixedsolution of ethyl acetate and hexane and the aqueous ethanol solutionare particularly preferable. More specifically, the mixed solution ofethyl acetate and hexane typically contains ethyl acetate and hexane ata volume ratio of 10:1 to 1:10. The ethanol concentration of the aqueousethanol solution used for the recrystallization is typically 30 to 90vol. %, preferably 40 to 70 vol. %. By carrying out therecrystallization using the mixed solution of ethyl acetate and hexaneor an aqueous ethanol solution adjusted to the foregoing concentration,the purity of the resultant equol further increases.

The recrystallization in Step 2-3 can be carried out using a generalmethod. For example, a solvent for recrystallization is added to theresidue resulted from the removal of solvent from the equol-containingfraction obtained in Step 2-2, and the residue is dissolved in thesolvent by heating. The solution is then cooled to precipitate equolcrystals. To further facilitate or accelerate equol crystallization,equol crystal seeds may be inoculated in the sample.

The temperature condition in the crystallization is determined accordingto the boiling point and the freezing point of the solvent. Generally,the residue is dissolved at about the boiling point of the solvent, andcrystals are precipitated at about 0 to 25° C.

3. Food Material

The present invention further provides a food material in which theequol-containing fermented soybean hypocotyl or its extract is dispersedin cacao mass. The following describes the details, such as theingredients, of the food material of the present invention.

Equol-Containing Fermented Soybean Hypocotyl and Extraction Thereof

The equol-containing fermented soybean hypocotyl used for the foodmaterial of the present invention is the same as the equol-containingfermented soybean hypocotyl described above in the section “1.Production Method for Equol-Containing Extract”. The equol-containingfermented soybean hypocotyl may be used for the food material of thepresent invention immediately after the fermentation, or after driedinto a dry solid as required. To improve the conservation stability ofthe equol-containing fermented soybean hypocotyl, it is more preferableto solidify the equol-containing fermented soybean hypocotyl by heatedair-drying or the like. The air-dried equol-containing fermented soybeanhypocotyl may be further processed into powder as required.

The extract of the equol-containing fermented soybean hypocotyldesignates an equol-containing extract resulted from solvent extractionwith respect to the equol-containing fermented soybean hypocotyl.

The extract of the equol-containing fermented soybean hypocotyl is notparticularly limited; however, the extract is preferably theequol-containing extract obtained by the method described above in thesection “1. Production Method for Equal-Containing Extract”, morepreferably the equol-containing extract obtained by Production Method Idescribed above. The extract of the equol-containing fermented soybeanhypocotyl of the present invention may be used in a liquid state;however, it is more preferable to solidify the liquid extract byremoving the extractant.

The food material of the present invention may contain either of theequol-containing fermented soybean hypocotyl or the extract thereof;however, the equol-containing fermented soybean hypocotyl isnutritionally preferred.

Cacao Mass

Cacao mass is a food material mainly used as an ingredient of chocolate.The cacao mass is obtained by processing cacao beans with fermentation,torrefaction, and grinding. The cacao mass used as a food material ofthe present invention is not particularly limited, as long as its safetyas a food material is approved.

Proportion of Equol-containing Fermented Soybean Hypocotyl or ExtractThereof and Cacao Mass

In the food material of the present invention, the proportion of theequol-containing fermented soybean hypocotyl or extract thereof andcacao mass is not limited; however, in order to improve the flavor ofthe food more preferably, the proportion of cacao mass is generally 10to 2000 parts by weight (dry weight), preferably 20 to 900 parts byweight, more preferably 20 to 400 parts by weight, based on 100 parts byweight of the equol-containing fermented soybean hypocotyl or extractthereof. Particularly, when using the equol-containing fermented soybeanhypocotyl, the proportion of cacao mass is generally 100 to 2000 partsby weight (dry weight), preferably 200 to 900 parts by weight, and morepreferably 200 to 400 parts by weight, based on 100 parts by weight ofthe equol-containing fermented soybean hypocotyl or extract thereof.

The food material of the present invention may consist only of theequol-containing fermented soybean hypocotyl or extract thereof andcacao mass, but may also contain other ingredients as detailed below.The sum proportion of the equol-containing fermented soybean hypocotylor extract thereof and cacao mass is 40 to 100 wt. %, preferably 60 to95 wt. %, and more preferably 70 to 95 wt. %, based on the gross amountof the food material of the present invention. By thus adjusting theproportion of the ingredients, the useful bioactivity derived from theequol-containing fermented soybean hypocotyl can be effectivelyexpressed, and the food will have a good flavor.

Arbitrary Ingredients

Insofar as the effects of the invention are not impaired, the foodmaterial of the present invention may contain various ingredients inaddition to the (a) equol-containing fermented soybean hypocotyl orextract thereof, and (b) cacao mass.

Examples of suitable ingredients for the food material of the presentinvention include sweeteners such as sugar, trehalose, acesulfam K,xylitol, scralose, erythritol, aspartame and starch syrup; oils and fatssuch as cacao butter or vegetable oils; emulsifiers such as soybeanlecithin; perfume; perfume preparations; milk; and water.

Form of Food Material

The shape of the food material of the present invention is notparticularly limited, and may be a form of powder, granule, chip, orplate, depending on the target product form of the food. To make gooduse of the texture characteristic of the food material of the presentinvention in the product, the food material is preferably processed inthe form of a granule or a chip, particularly preferably a granule or achip of about 30 to 1000 mg per piece.

Production Method for Food Material of the Present Invention

The food material of the present invention is prepared by mixing theequol-containing fermented soybean hypocotyl or extract thereof, cacaomass, and other necessary ingredients at a temperature where the cacaobutter in the cacao mass dissolves (preferably 30 to 50° C.), and thencooling and solidifying the mixture. As a result, the food material ofthe present invention in which the equol-containing fermented soybeanhypocotyl is dispersed in cacao mass is prepared. As required, the foodmaterial of the present invention is subjected to pulverization,trituration, sizing or the like to be formed into a desired shape.Alternatively, the food material of the present invention may be shapedusing extrusion granulation during the production.

Use of Food Material

The food material of the present invention may be incorporated intovarious kinds of foodstuffs, as a raw material of an equol-containingfood, or as a food additive. The present invention also provides suchequol-containing foods made of the above-described food material.

The equol-containing food of the present invention is not particularlylimited, and includes baked confectioneries and frozen desserts.

Examples of the baked confectioneries include various glutinous ricecrackers; wheat crackers such as wafers or waffles; biscuits; cookies;soda crackers; pies; cakes; doughnuts; and bread. Examples of the frozendesserts include ice cream, shaved ice, and ice pops.

Among them, baked confectioneries are one of the suitable forms ofequol-containing food. Particularly, a baked confectionery made ofsoybean powder contains not only equol but also other soybean-deriveduseful components; therefore, the baked confectionery is highly useful,providing a superior health maintenance effect.

One preferred example of the baked confectionery made of soybean powdercontains soybean powder, egg, and butter, but no wheat. The soybeanpowder to be used for this type of cookie is preferably processed byheating or the like to deactivate or diminish the lipoxygenases and toremove or reduce the soybean smell. Such a baked confectionery can beproduced by, for example, using 0 to 35 parts by weight of egg, 10 to 65parts by weight of butter, 0.1 to 50 parts by weight of the foodmaterial of the present invention, and other ingredients, per 100 partsby weight of soybean powder. These ingredients are mixed with anappropriate amount of water to make the dough. The dough is then formedinto a predetermined shape, and baked at about 60 to 250° C. for 2 to 60minutes.

The proportion of the food material of the present invention in theequol-containing food is not particularly limited, and is adjusteddepending on the equol content in the food material of the presentinvention, the form of the equol-containing food or the like. Theproportion of the food material of the present invention is generally 3to 30 wt. %, preferably 5 to 20 wt. %, and more preferably 5 to 8 wt. %,based on the total amount of the materials of the equol-containing food.

Further, the proportion of equol in the equol-containing food isgenerally 0.002 to 0.1 wt. %, preferably 0.004 to 0.05 wt. %, and morepreferably 0.005 to 0.03 wt. %, based on the total amount of thematerials of the equol-containing food. By ensuring this proportion ofthe food material of the present invention, the useful bioactivityderived from the equol-containing fermented soybean hypocotyl isexpressed in the food, while maintaining the good flavor of theequol-containing food.

The equol-containing food is produced by mixing the food material of thepresent invention with a predetermined amount of the other ingredients,and subjecting the mixture to forming, baking, cooling etc. depending onthe target form of the food product. The equol-containing food is madeof an equol-containing fermented soybean hypocotyl, and contains varioususeful bioactivity materials including equol, thereby expressing variousbioactivities or pharmacological activities. Therefore, theequol-containing food is useful not only as a general food but also as afood for specified health use, nutritional supplement, functional food,invalid food or the like. The food containing the fermented soybeanhypocotyl of the present invention is particularly useful as anutraceutical product.

For example, the equol-containing food has an effect of preventing ortreating various diseases or symptoms including menopausal disorders,osteoporosis, prostatic hypertrophy, and metabolic syndrome; or forblood cholesterol level reduction, skin-whitening, pimples, intestinalcontrol, obesity, and diuresis. Particularly, the equol-containing foodis suitable for prevention or treatment of indefinite complaint orpost-menopausal symptoms (e.g., osteoporosis, menopausal disorder) ofmiddle-aged women.

The intake of the equol-containing food per day is determined dependingon the equol content in the food, age, weight, symptom of the consumer,or the dosage number.

4. Foods of Various Forms

The present invention further provides various forms of food containingthe equol-containing fermented soybean hypocotyl or extract thereof.Examples of the foods of the present invention includes beverages,dietary supplements, creamy foods, desserts, confectioneries,seasonings, retort-packed foods, processed meat products, fish pastes,processed egg products, canned foods, breads, frozen desserts, processedsoybean foods, cooked rice products, and soup.

The following explains in detail the equol-containing fermented soybeanhypocotyl and the extract thereof, and each form of food provided by thepresent invention.

Equol-Containing Fermented Soybean Hypocotyl and Extract Thereof

The equol-containing fermented soybean hypocotyl or extract thereof usedfor the present invention is the same as that used in the section “3.Food Material”.

The food of the present invention may contain any of the items in theforegoing list of equol-containing fermented soybean hypocotyls orextracts thereof. The food of the present invention preferably containsan equol-containing fermented soybean hypocotyl.

Forms of Food

The equol-containing fermented soybean hypocotyl or extract thereof iseffective in preventing or treating various diseases or symptomsincluding menopausal disorders, osteoporosis, prostatic hypertrophy, andmetabolic syndrome; or for blood cholesterol level reduction,skin-whitening, pimples, intestinal control, obesity, and diuresis.Particularly, the equol-containing food is suitable for the preventionor treatment of indefinite complaints or post menopausal symptoms (e.g.osteoporosis, menopausal disorders) of middle-aged women. Accordingly,the various forms of food of the present invention may be provided asfunctional foods effective for those diseases or symptoms. Morespecifically, the food of the present invention containing fermentedsoybean hypocotyl is not only useful as a general food, but also usefulas a food for specified health use, a nutritional supplement, afunctional food, an invalid diet food or the like. The food of thepresent invention containing fermented soybean hypocotyl is particularlyuseful as a nutritional supplement.

The following explains specific examples of the food forms of thepresent invention.

(1) Beverage

The present invention provides a beverage containing theequol-containing fermented soybean hypocotyl or extract thereof.

The beverage of the present invention may be any kind of beverage;however, it is suitable for beverages containing fruit juice; beveragescontaining vegetable juice; carbonated beverages such as sparklingwater, cola, a fruit-juice-containing carbonated beverage, ginger ale,soda; soft drinks such as a sports drink; coffee beverages such ascoffee or milk coffee; teas such as English-style tea, green tea, oroolong tea; cocoa; lactic acid bacteria beverages; milk beverages suchas cow's milk, fruit-flavored milk, or sour milk beverages; alcoholicbeverages such as beer, shochu (distilled spirit), whiskey, cocktails,or shochu mixed with soda water.

The content of the equol-containing fermented soybean hypocotyl orextract thereof in each beverage of the present invention is notlimited, and is individually determined according to the equol contentin the fermented soybean hypocotyl or extract thereof, the type ofbeverage, the expected effect or the like. For example, the content ofthe fermented soybean hypocotyl or extract thereof in the beverage isgenerally 0.02 to 15 wt. %, preferably 0.1 to 1 wt. %, and morepreferably 0.2 to 0.5 wt. % (dry weight).

(2) Dietary Supplement

The present invention provides a dietary supplement containing theequol-containing fermented soybean hypocotyl or extract thereof.

The form of the dietary supplement of the present invention is notlimited, and includes a softgel, a tablet, powder or the like, amongwhich a softgel is particularly preferred. The softgel or powder ispreferably contained in a capsule at a predetermined amount.

One of the suitable embodiments of the softgel of the present inventionis a softgel that contains gelatin, water and glycerin having theequol-containing fermented soybean hypocotyl or extract therein. Thesoftgel may further contain other additives such as soybean oil,beeswax, lecithin, or β-carotin.

The content of the equol-containing fermented soybean hypocotyl orextract thereof in each dietary supplement of the present invention isnot limited, and is individually determined according to the equolcontent in the fermented soybean hypocotyl or extract thereof, the typeof supplement, the expected effect or the like. For example, the contentof the fermented soybean hypocotyl or extract thereof in the dietarysupplement is 2 to 100 wt. % (dry weight).

(3) Creamy Food

The present invention provides a creamy food containing theequol-containing fermented soybean hypocotyl or extract thereof.

The creamy food of the present invention is not particularly limited andmay include whipped cream, custard cream, butter cream, fresh cream, andcoffee whitener.

The content of the equol-containing fermented soybean hypocotyl orextract thereof in each creamy food of the present invention is notlimited, and is individually determined according to the equol contentin the fermented soybean hypocotyl or extract thereof, the type ofcreamy food, the expected effect or the like. For example, the contentof the fermented soybean hypocotyl or extract thereof in the creamy foodis generally 0.03 to 5 wt. %, preferably 0.15 to 1 wt. %, and morepreferably 0.3 to 0.5 wt. % (dry weight).

(4) Dessert

The present invention provides a dessert containing the equol-containingfermented soybean hypocotyl or extract thereof.

The dessert is not particularly limited and may include yoghurt, jelly,pudding, mousse or the like.

The content of the equol-containing fermented soybean hypocotyl orextract thereof in each dessert of the present invention is not limited,and is individually determined according to the equol content in thefermented soybean hypocotyl or extract thereof, the type of dessert, theexpected effect or the like. For example, the content of the fermentedsoybean hypocotyl or extract thereof in the dessert is generally 0.03 to5 wt. %, preferably 0.15 to 1 wt. %, and more preferably 0.3 to 0.5 wt.% (dry weight).

(5) Snack

The present invention provides a snack containing the equol-containingfermented soybean hypocotyl or extract thereof.

The snack is not particularly limited and may include any western orJapanese sweets including baked or steamed confectionery. Examples ofthe snack of the present invention include candies, lollies, caramels,pretzels, tablets, gums, chocolates, cookies, bean confectionery, wafer,cheese crackers, gummy candies, ordinary rice crackers, and Suhama(Japanese traditional confectionery), dry confectionery, and Yokan(azuki bean jelly), steamed bean-jam bun, bracken-starch dumplings,pound cakes, apple pie fillings, savory snacks, and glutinous ricecrackers. Among them, one of the suitable embodiments of the snack is abaked confectionery containing soy flour.

The content of the equol-containing fermented soybean hypocotyl orextract thereof in each snack of the present invention is not limited,and is individually determined according to the equol content in thefermented soybean hypocotyl or extract thereof, the type of snack, theexpected effect or the like. For example, the content of the fermentedsoybean hypocotyl or extract thereof in the snack is generally 0.01 to99 wt. %, preferably 0.04 to 36 wt. %, and more preferably 0.08 to 18wt. % (dry weight).

(6) Seasoning

The present invention provides a seasoning containing theequol-containing fermented soybean hypocotyl or extract thereof.

The seasoning is not particularly limited and may include barbecuesauce, sauce for Kabayaki (broiled seafood), seasoning of squid-chinmi,ketchup, tsuyu (soup), dressing, sesame dipping sauce, snack seasoningsand soy sauce.

The content of the equol-containing fermented soybean hypocotyl orextract thereof in each seasoning of the present invention is notlimited, and is individually determined according to the equol contentin the fermented soybean hypocotyl or extract thereof, the type ofseasoning, the expected effect or the like. For example, the content ofthe fermented soybean hypocotyl or extract thereof in the seasoning isgenerally 0.04 to 40 wt. %, preferably 0.2 to 8 wt. %, and morepreferably 0.4 to 4 wt. % (dry weight).

(7) Retort-Packed Food

The present invention provides a retort-packed food (retort pouch food)containing the equol-containing fermented soybean hypocotyl or extractthereof.

The retort-packed food is not particularly limited and may includevarious seasoning materials including a sauce premix for curry sauce,hashed rice sauce, stew, meat sauce, or white sauce; a premix forChinese-style donburi, beef donburi, and chicken and egg donburi; apremix for Ma-po Dou-fu (Tofu with chili and bean-based sauce), and apremix for Kamameshi (a Japanese rice dish cooked with various kinds ofmeat, seafood, and vegetables).

The content of the equol-containing fermented soybean hypocotyl orextract thereof in each retort-packed food of the present invention isnot limited, and is individually determined according to the equolcontent in the fermented soybean hypocotyl or extract thereof, the typeof retort-packed food, the expected effect or the like. For example, thecontent of the fermented soybean hypocotyl or extract thereof in theretort-packed food is generally 0.003 to 4 wt. %, preferably 0.01 to 0.8wt. %, and more preferably 0.03 to 0.4 wt. % (dry weight).

(8) Processed Meat Product

The present invention provides a processed meat product containing theequol-containing fermented soybean hypocotyl or extract thereof.

The processed meat product of the present invention is not particularlylimited and may include sausages (dry, semi-dry, bologna, wiener,smoke), liver paste, bacon, shoulder bacon, ham, hamburger steak, andmeatballs.

The content of the equol-containing fermented soybean hypocotyl orextract thereof in each processed meat product of the present inventionis not limited, and is individually determined according to the equolcontent in the fermented soybean hypocotyl or extract thereof, the typeof processed meat product, the expected effect or the like. For example,the content of the fermented soybean hypocotyl or extract thereof in theprocessed meat product is generally 0.02 to 0.4 wt. %, preferably 0.1 to0.8 wt. %, and more preferably 0.2 to 0.4 wt. % (dry weight).

(9) Fish Paste Product

The present invention provides a fish paste product containing theequol-containing fermented soybean hypocotyl or extract thereof.

The fish paste product of the present invention is not particularlylimited and may include boiled fish paste and fishcake tubes.

The content of the equol-containing fermented soybean hypocotyl orextract thereof in each fish paste product of the present invention isnot limited, and is individually determined according to the equolcontent in the fermented soybean hypocotyl or extract thereof, the typeof fish paste product, the expected effect or the like. For example, thecontent of the fermented soybean hypocotyl or extract thereof in thefish paste product is generally 0.03 to 5 wt. %, preferably 0.1 to 1 wt.%, and more preferably 0.3 to 0.5 wt. % (dry weight).

(10) Processed Egg Product

The present invention provides a processed egg product containing theequol-containing fermented soybean hypocotyl or extract thereof.

The processed egg product of the present invention is not particularlylimited and may include Japanese rolled omelet, thick Japanese omelet,western-style omelet, and scrambled eggs.

The content of the equol-containing fermented soybean hypocotyl orextract thereof in each processed egg product of the present inventionis not limited, and is individually determined according to the equolcontent in the fermented soybean hypocotyl or extract thereof, the typeof processed egg product, the expected effect or the like. For example,the content of the fermented soybean hypocotyl or extract thereof in theprocessed egg product is generally 0.03 to 5 wt. %, preferably 0.1 to 1wt. %, and more preferably 0.3 to 0.5 wt. % (dry weight).

(11) Canned Food

The present invention provides a canned food containing theequol-containing fermented soybean hypocotyl or extract thereof.

The canned food of the present invention is not particularly limited andmay include canned fruit such as canned oranges or canned peaches;canned seafood such as canned tuna or canned sardines; canned jam;canned soup; and canned sauce.

The content of the equol-containing fermented soybean hypocotyl orextract thereof in each canned food of the present invention is notlimited, and is individually determined according to the equol contentin the fermented soybean hypocotyl or extract thereof, the type ofcanned food, the expected effect or the like. For example, the contentof the fermented soybean hypocotyl or extract thereof in the canned foodis generally 0.03 to 5 wt. %, preferably 0.1 to 1 wt. %, and morepreferably 0.3 to 0.5 wt. % (dry weight).

(12) Bread

The present invention provides bread containing the equol-containingfermented soybean hypocotyl or extract thereof.

The bread of the present invention is not particularly limited and mayinclude a loaf of bread, French bread, stuffed bread, melon bread,bean-jam bread, cream bread, jam bread, bread rolls, and croissants.

The content of the equol-containing fermented soybean hypocotyl orextract thereof in each bread of the present invention is not limited,and is individually determined according to the equol content in thefermented soybean hypocotyl or extract thereof, the type of bread, theexpected effect or the like. For example, the content of the fermentedsoybean hypocotyl or extract thereof in the bread is generally 0.01 to50 wt. %, preferably 0.05 to 0.6 wt. %, and more preferably 0.1 to 0.3wt. % (dry weight).

(13) Frozen Dessert

The present invention provides a frozen dessert containing theequol-containing fermented soybean hypocotyl or extract thereof.

The bread of the present invention is not particularly limited and mayinclude ice cream and sherbet.

The content of the equol-containing fermented soybean hypocotyl orextract thereof in each frozen dessert of the present invention is notlimited, and is individually determined according to the equol contentin the fermented soybean hypocotyl or extract thereof, the type offrozen dessert, the expected effect or the like. For example, thecontent of the fermented soybean hypocotyl or extract thereof in thefrozen dessert is generally 0.03 to 5 wt. %, preferably 0.1 to 1 wt. %,and more preferably 0.3 to 0.5 wt. % (dry weight).

(14) Processed Soybean Food

The present invention provides a processed soybean food containing theequol-containing fermented soybean hypocotyl or extract thereof.

The processed soybean food of the present invention is not particularlylimited and may include tofu, thick deep-fried tofu, fried tofu mixedwith vegetables, soy milk skin, fermented soybeans, and soy milk.

The content of the equol-containing fermented soybean hypocotyl orextract thereof in each processed soybean food of the present inventionis not limited, and is individually determined according to the equolcontent in the fermented soybean hypocotyl or extract thereof, the typeof processed soybean food, the expected effect or the like. For example,the content of the fermented soybean hypocotyl or extract thereof in theprocessed soybean food is generally 0.03 to 0.5 wt. %, preferably 0.1-1wt. %, and more preferably 0.3-0.5 wt. % (dry weight).

(15) Cooked Rice Product

The present invention provides a cooked rice product containing theequol-containing fermented soybean hypocotyl or extract thereof. Thecooked rice product of the present invention is not particularly limitedand may include fried rice, pilaf, rice gruel, steamed glutinous ricewith chestnut, cooked white rice, red beans rice, and rice steamed withmeat and vegetables.

The content of the equol-containing fermented soybean hypocotyl orextract thereof in each cooked rice product of the present invention isnot limited, and is individually determined according to the equolcontent in the fermented soybean hypocotyl or extract thereof, the typeof cooked rice product, the expected effect or the like. For example,the content of the fermented soybean hypocotyl or extract thereof in thecooked rice product is generally 0.01 to 2.5 wt. %, preferably 0.02 to0.5 wt. %, and more preferably 0.04 to 0.25 wt. % (dry weight).

(16) Soup

The present invention provides soup containing the equol-containingfermented soybean hypocotyl or extract thereof.

The soup of the present invention is not particularly limited and mayinclude corn cream soup, consomme, minestrone, onion soup, and potage.

The content of the equol-containing fermented soybean hypocotyl orextract thereof in each soup of the present invention is not limited,and is individually determined according to the equol content in thefermented soybean hypocotyl or extract thereof, the type of soup, theexpected effect or the like. For example, the content of the fermentedsoybean hypocotyl or extract thereof in the soup is generally 0.02 to 15wt. %, preferably 0.1 to 1 wt. %, and more preferably 0.2 to 0.5 wt. %(dry weight).

EXAMPLES

The following more specifically explains the present invention withreference to Reference Examples, Examples etc. The present invention ishowever not limited to these examples.

Reference Examples 1-1 to 1-3 Production of Equol-Containing FermentedSoybean Hypocotyl

Soybean hypocotyl powder, arginine, and water were mixed to prepare asoybean hypocotyl solution (material) having a composition as shown inTable 1. A Lactococcus 20-92 strain (FERM BP-10036) was inoculated inthis soybean hypocotyl solution of 5 ml, and the sample was subjected tostationary culture for 96 hours at 37° C. under anaerobic conditions.After that, the resulting fermentation solution (culture solution) wassterilized by heating for a minute at 100° C., followed by drying at 80°C. The dried product was processed into a powder using a homogenizer toobtain fermented soybean hypocotyl powder.

Table 1 shows the viable cell count and pH of the culture after 96 hoursculture, the amount of fermented soybean hypocotyl powder collected andthe equol content of the fermented soybean hypocotyl powder. From thedata, it is shown that fermentation of soybean hypocotyl powder usingequol-producing bacteria produces equol with high efficiency.

TABLE 1 Ref. Ex. 1-1 Ref. Ex. 1-2 Ref. Ex. 1-3 Composition Soybean 0.25g 0.5 g 0.75 g of Soybean Hypocotyl Hypocotyl Powder (dry Solution wt.)(Material) Arginine 0.005 g 0.005 g 0.005 g Water AppropriateAppropriate Appropriate Quantity Quantity Quantity Total Amount 5 ml 5ml 5 ml PH 6.75 ± 0.03 6.54 ± 0.02 6.39 ± 0.03 Analytical Viable  7.9 ±0.1  8.2 ± 0.1  8.3 ± 0.2 Result of Bacterial Fermented Counts of LiquidFermented Liquid (log cfu/ml) PH of 7.00 ± 0.03 6.88 ± 0.01 6.76 ± 0.02Fermented Liquid Analysis of Equol 3.85 mg (—) 3.44 mg (—) 5.38 mgComposition (48.9 wt. of %) Fermented Daidzein n.d. (—) n.d. 1.18 mgSoybean Compounds (10.7 wt. %) Hypocotyl Genistein n.d. (—) n.d. 1.45 mgPowder in Compounds (13.2 wt. terms of %) Isoflavone Glycitein n.d. (—)n.d. 3.00 mg Compounds (27.2 wt. %)

Each Example was performed using three lots of soybean hypocotyl powder(N=3). In the analysis of isoflavone composition in the table, the valueon the left denotes a content (mg) of each isoflavone per gram of thefermented soybean hypocotyl, and the value in the bracket on the rightdenotes the ratio (wt. %) of each isoflavone to the gross isoflavoneamount (100 wt. %) in the fermented soybean hypocotyl. “n.d.” indicatesthat the measurement is not done, and “−” in the bracket indicates thatthe calculation is not done.

Reference Example 1-4 Production of Equol-Containing Fermented SoybeanHypocotyl

A Lactococcus 20-92 strain (FERM BP-10036) was inoculated to a 5 mlsoybean hypocotyl solution containing 10 wt of soybean hypocotyl powderand 0.1 wt. % of arginine, and the sample was subjected to fermentationby carrying out stationary culture for 96 hours at 37° C. underanaerobic conditions. After that, the resulting fermentation solution(culture solution) was sterilized by heating for a minute at 100° C.,followed by drying at 80° C. The dried product was processed into powderusing a homogenizer to obtain fermented soybean hypocotyl powder.

The respective components of the soybean hypocotyl powder(“Pre-fermentation” in Table 2 and Table 3) and of the fermented soybeanhypocotyl powder (“Post-fermentation” in Table 2 and Table 3) wereanalyzed. Table 2 shows an analysis regarding soy isoflavone, and Table3 shows an analysis regarding nutritional components. These tables showthat the fermented soybean hypocotyl obtained by the fermentation ofsoybean hypocotyl using a Lactococcus 20-92 strain has a high equolcontent. Further, oligosaccharide content for such oligosaccharides asraffinose or stachyose remains substantially the same afterfermentation, which indicates that the fermentation did not influencethe oligosaccharide content. Meanwhile, the tables show that argininewas converted into ornithine by the fermentation. Accordingly, by addingarginine to the soybean hypocotyl before fermentation using aLactococcus 20-92 strain, the resulting fermented substance contains notonly equol but also ornithine.

TABLE 2 Soybean Isoflavones Per 100 g Component Pre-fermentationPost-fermentation Equol N.D. 632.0 mg Daidzin 566.4 mg  29.7 mgMalonyldaidzin 124.9 mg N.D. Acetyldaidzin 364.8 mg  25.4 mg Daidzein 7.1 mg  24.4 mg Dihydrodaidzein N.D.  49.4 mg Genistin 111.7 mg  3.2 mgMalonylgenistin  35.1 mg N.D. Acetylgenistin 146.1 mg  3.7 mg Genistein 0.9 mg  22.5 mg Dihydrogenistein N.D. 112.0 mg Glycitin 331.7 mg  53.6mg Malonylglycitin  65.0 mg N.D. Acetylglycitin 169.2 mg  34.8 mgGlycitein  19.1 mg 292.3 mg Dihydroglycitein N.D.  8.2 mg TotalIsoflavones 1942.0 mg  1291.2 mg  N.D. refers to “Not Detected”

TABLE 3 Nutritional Component Per 100 g Component Pre-fermentationPost-fermentation Moisture 3.2 g 6.2 g Protein 38.1 g 38.3 g Fat 13.0 g14.5 g Ash 4.3 g 4.0 g Saccharide 30.9 g 26.8 g Dietary Fiber 10.5 g10.2 g Energy 414 kcal 411 kcal Sucrose 7.95 g 7.42 g Raffinose 1.37 g1.34 g Stachyose 9.04 g 8.38 g Trans Fatty Acids N.D. N.D.Phospholipids(as stear-, ole-, 3.33 g 2.92 g and lecithin) Free Arginine881 mg 12 mg Free Ornithine N.D. 1.06 g Soyasapogenol A N.D. N.D.Soyasapogenol B N.D. N.D. Soybean Saponin 3.6 g 3.8 g N.D. refers to“Not Detected”

Reference Examples 1-5 to 1-11 Production of Equol-Containing FermentedSoybean Hypocotyl

In these examples, fermented soybean hypocotyl powder (ReferenceExamples 1-5 to 1-11) were produced in the same manner as in ReferenceExamples 1-3 except that seven lots of soybean hypocotyl powderdifferent from the three lots of Reference Example 1-3 were used. Thecompositions of isoflavone in the obtained fermented soybean hypocotylwas analyzed as shown in Table 4. This result also shows that each ofthe fermented soybean hypocotyls of Reference Examples 1-5 to 1-11 has ahigh equol content with isoflavone proportion that was not attained byany known arts.

TABLE 4 Isoflavone Proportions Daidzein Genistein Glycitein EquolCompounds Compounds Compounds Ref. 6.51 mg 0.71 mg 0.53 mg 2.71 mg Ex.(62.2 wt. %) (6.8 wt. %) (5.1 wt. %) (25.9 wt. %) 1-5 Ref. 6.25 mg 0.48mg 0.35 mg 3.12 mg Ex. (61.3 wt. %) (4.7 wt. %) (3.4 wt. %) (30.6 wt. %)1-6 Ref. 5.38 mg 1.18 mg 1.45 mg 3.00 mg Ex. (48.9 wt. %) (10.7 wt. %)(13.2 wt. %) (27.2 wt. %) 1-7 Ref. 6.43 mg 0.61 mg 0.48 mg 2.62 mg Ex.(63.4 wt. %) (6.0 wt. %) (4.7 wt. %) (25.8 wt. %) 1-8 Ref. 6.05 mg 0.51mg 0.30 mg 2.57 mg Ex. (64.2 wt. %) (5.4 wt. %) (3.2 wt. %) (27.3 wt. %)1-9 Ref. 6.11 mg 0.37 mg 0.10 mg 2.74 mg Ex. (65.6 wt. %) (4.0 wt. %)(1.1 wt. %) (29.4 wt. %) 1-10 Ref. 6.3 mg 0.49 mg 0.37 mg 3.19 mg Ex.(60.9 wt. %) (4.73 wt. %) (3.6 wt. %) (30.8 wt. %) 1-11

The value in the upper column denotes a content (mg) of each isoflavoneper gram of the fermented soybean hypocotyl, and the value in the lowercolumn denotes the ratio (wt. %) of each isoflavone to the grossisoflavone amount (100 wt. %) in the fermented soybean hypocotyl.

Reference Experiment Example 1 Allergen Confirmation Test

Soybean hypocotyl is known to contain allergens including Gym4, Gm30K,Gm28K, 7S globulin mix (β-conglycine), oleosin, and trypsin inhibitors.The existence of allergens in the equol-containing fermented soybeanhypocotyl produced in Reference Example 1-1 was examined using thefollowing test.

First, an appropriate amount of the equol-containing fermented soybeanhypocotyl obtained in Reference Example 1-1 was added to an extractionbuffer (Tris HCl, pH 7.5, containing 1M EDTA and an appropriate amountof protease inhibitor). The sample was stirred well to extract awater-soluble component. The resulting solution was filtered to removethe solid content to obtain a liquid extract. All proteins contained inthis liquid extract were detected using a protein assay system(Bio-Rad). Further, the major allergens (Gym4, Gm30K, Gm28K, 7S globulinmix, oleosin, and trypsin inhibitor) contained in the liquid extractwere detected using western blotting. Further, for comparison, thedetection of total protein and allergen was carried out in the samemanner using soybean cotyledon powder and soybean hypocotyl powder,instead of the fermented soybean hypocotyl.

FIGS. 1 to 3 show the result. FIG. 1 shows the result for the detectionof total protein; FIG. 2 shows the result for the detection of Gym4,Gm30K, and Gm28K; and FIG. 3 shows the result for the detection of 7Sglobulin mix, oleosin, and trypsin inhibitor.

These results show that the major allergen content of a soybean or asoybean hypocotyl is reduced in the equol-containing fermented soybeanhypocotyl.

Example 1 Production of Equol-Containing Extract Example 1-1 Productionof Equol-Containing Extract by Production Method I

One gram of the equol-containing fermented soybean hypocotyl powderproduced under the same conditions as in Reference Examples 1 to 3 wasadded to 20 ml of the extractant according to Table 5; and 120 minutesof shaking was performed at room temperature. Thereafter, the residue(“Residue 1”, hereinafter) was removed by centrifugation to isolate aliquid extract (“Liquid Extract 1-1”, hereinafter). Next, Liquid Extract1-1 was evaporated to dryness using an evaporator, 5 ml of ethanol wasadded thereto, and 10 minutes of shaking was performed at roomtemperature. Thereafter, the residue (“Residue 2”, hereinafter) wasremoved by centrifugation to isolate a liquid extract (“Liquid Extract1-2”, hereinafter). Liquid Extract 1-2 was then evaporated to drynessusing an evaporator to obtain a solid extract.

Further, another 20 ml of extractant of the same composition was addedto Residue 1, and 120 minutes shaking was performed at room temperature.Thereafter, the residue was removed by centrifugation to isolate aliquid extract (Liquid Extract 2-1, hereinafter). Liquid Extract 2-1 wasthen evaporated to dryness using an evaporator. 5 ml of ethanol wasadded thereto, and 10 minutes of shaking was performed at roomtemperature. Thereafter, the residue was removed by centrifugation toisolate an extract (“Liquid Extract 2-2”, hereinafter). Liquid Extract2-2 was evaporated to dryness using an evaporator to obtain a solidextract.

TABLE 5 Name of Extractant Composition of Extractant 25Et EthanolSolution containing 25 vol. % of Ethanol and 75 vol. % of Purified Water50Et Ethanol Solution containing 50 vol. % of Ethanol and 50 vol. % ofPurified Water 75Et Ethanol Solution containing 75 vol. % of Ethanol and25 vol. % of Purified Water 100Et  100 vol. % of Ethanol

Liquid Extracts 1-1 and 1-2, Liquid Extracts 2-1 and 2-2, and Residue 2were subjected to thin-layer chromatography (TLC) analysis (as a mobilephase, hexane:chloroform:ethyl acetate=1:1:1 (volume ratio) was used forequol detection; and ethyl acetate:propanol:chloroform:methanol:0.9%KCl=25:25:25:10:9 (volume ratio) was used for saponin detection) so asto measure saponin content and equol content. The weight of the solidextract obtained from Liquid Extract 1-2 and its equol concentrationwere measured. FIGS. 4 to 7 and Table 6 show the results. FIG. 4 showsthe results of TLC analysis of the equol contents in Liquid Extracts 1-2and 2-2; FIG. 5 shows the results of TLC analysis of the saponincontents in Liquid Extracts 1-2 and 2-2; FIG. 6 shows the results of TLCanalysis of the equol contents in Liquid Extract 1-2 and Residue 2; andFIG. 7 shows the results of TLC analysis of the saponin contents inLiquid Extract 1-2 and Residue 2.

According to the figures, it was found that when the equol-containingfermented soybean hypocotyl was subjected to the extraction usingethanol, the extract contained a large amount of oil, and the equolrecovery rate was low.

In contrast, when the equol-containing fermented soybean hypocotyl wassubjected to the extraction using an aqueous ethanol, the inclusion ofoil was suppressed, and the equol recovery rate increased. Particularly,it became clear that use of an aqueous ethanol solution having anethanol concentration of 75 vol. % significantly increases the equolcollection rate. Further, it was also found that extraction using a 75vol. % ethanol minimizes the extraction of fat components. Furthermore,according to the fact that saponin in a concentrated amount was detectedin Residue 2, saponin can be removed without reducing the equol amountby subjecting the residue, which was obtained by removing solvent fromLiquid Extract 1-1, to extraction using ethanol.

TABLE 6 EQL-containing Fermented Soybean Hypocotyl Solid ExtractCollected from Liquid Extract 1-2 Amount Used EQL EQL Dry EQL EQL EQLEQL Extract for Content Concentration Weight Content ConcentrationCollection Concentration Type Extraction (g) (mg) (wt. %) (mg) (mg) (wt.%) Rate (%) Rate 25Et 1 5.38 0.54 43 1.78 4.14 33.1 7.7 50Et 1 5.38 0.5436 2.68 7.44 49.8 13.8 75Et 1 5.38 0.54 121 5.56 4.60 103.4 8.5 100Et  15.38 0.54 146 1.50 1.03 27.9 1.9

EQL represents equol. The “concentration rate” represents the EQLconcentration rate of the solid extract when assuming the EQLconcentration in the EQL-containing fermented soybean hypocotyl is 1.

Example 1-2 Production of Equol-Containing Extract by Production MethodI

In this example, equol-containing fermented soybean hypocotyl powder wasproduced under the same conditions as in Reference Example 1-3 exceptthat a different lot of soybean hypocotyl powder was used.

30 g of the equol-containing fermented soybean hypocotyl powder wasadded to 150 ml of a 70 vol. % aqueous ethanol solution (100 mlconsisting of ethanol and 30 ml of water) or 150 ml of a 90 vol. %aqueous ethanol solution (100 ml consisting of ethanol and 10 ml ofwater); and eight hours stirring was performed at room temperature.After removing the residue by filtration, the residue was washed with a20 ml of aqueous ethanol solution having the same composition as that ofthe extractant. The filtrate and the liquid after washing were collectedto obtain a liquid extract. The liquid extract was concentrated underreduced pressure to obtain a solid extract.

The weight of the obtained solid extract and the weight of equolcontained therein and the concentrations of daidzein, dihydrodaidzin,genistein, dihydrogenistin, glystein, dihydroglystein were measured, asshown in Tables 7 and 8.

TABLE 7 Extraction Results for 70 vol. % Ethanol Solution EQL-containingFermented Soybean Hypocotyl Collected Solid Extract Amount for DryWeight Weight Extraction (g) 30 (mg) 8000 EQL EQL Content EQL EQLContent EQL EQL Collection EQL (mg) Concentration (mg) ConcentrationRate (%) Concentration (wt. %) (wt. %) Rate 161.40  0.54 158.98  1.9898.5  3.7 DZN DZN Content DZN DZN Content DZN DZN Collection DZN (mg)Concentration (mg) Concentration Rate (%) Concentration (wt. %) (wt. %)Rate (%) 6.00 0.02 0.00 0.00 0.0 0.0 DHD DHD Content DHD DHD Content DHDDHD Collection DHD (mg) Concentration (mg) Concentration Rate (%)Concentration (wt. %) (wt. %) Rate (%) 9.00 0.03 0.09 0.00 0.00  0.00GNT GNT Content GNT GNT Content GNT GNT Collection GNT (mg)Concentration (mg) Concentration Rate (%) Concentration (wt. %) (wt. %)Rate (%) 6.90 0.02 0.31 0.00 4.5  0.2 DHG DHG Content DHG DHG ContentDHG DHG Collection DHG (mg) Concentration (mg) Concentration Rate (%)Concentration (wt. %) (wt. %) Rate (%) 32.7  0.11 6.96 0.09 21.3  0.5GLY GLY Content GLY GLY Content GLY GLY Collection GLY (mg)Concentration (mg) concentration Rate (%) Concentration (wt. %) (wt. %)Rate (%) 62.70  0.21 33.17  0.41 52.9  2.0 DHG DHG Content DHG DHGContent DHG DHG Collection DHG (mg) Concentration (mg) ConcentrationRate (%) Concentration (wt. %) (wt. %) Rate (%) 0.9  0.00 0.00 0.00 0.00 0.00

EQL represents equol; DZN represents daidzein; DHD representsdihydrodaidzin; GNT represents genistein; DHG representsdihydrogenistin; GLY represents glystein; and DHG representsdihydroglystein.

The isoflavone content (mg) denotes the gross weight of each isoflavonecontained in the EQL-containing fermented soybean hypocotyl (material:30 g) or in the obtained isolated extract (8000 mg); and the isoflavoneconcentration (wt. %) denotes the proportion of each isoflavonecontained in the EQL-containing fermented soybean hypocotyl (material)or in the obtained isolated extract. The “Concentration Rate” representsthe concentration rate of each component in the solid extract whenassuming the concentration of the component in the EQL-containingfermented soybean hypocotyl is 1.

TABLE 8 Extraction Results for 90 vol. % Ethanol Solution EQL-containingFermented Soybean Hypocotyl Collected Solid Extract Amount for DryWeight Weight Extraction (g) 30 (mg) 4100 EQL EQL Content EQL EQLContent EQL EQL Collection EQL (mg) Concentration (mg) ConcentrationRate (%) Concentration (wt. %) (wt. %) Rate 161.40  0.54 158.13  3.8998.0 7.2 DZN DZN Content DZN DZN Content DZN DZN Collection DZN (mg)Concentration (mg) Concentration Rate (%) Concentration (wt. %) (wt. %)Rate (%) 6.00 0.02 0.00 0.00  0.0 0.0 DHD DHD Content DHD DHD ContentDHD DHD Collection DHD (mg) Concentration (mg) Concentration Rate (%)Concentration (wt. %) (wt. %) Rate (%) 9.00 0.03 0.12 0.00  1.3 0.1 GNTGNT Content GNT GNT Content GNT GNT Collection GNT (mg) Concentration(mg) Concentration Rate (%) Concentration (wt. %) (wt. %) Rate (%) 6.900.02 0.34 0.01  4.9 0.4 DHG DHG Content DHG DHG Content DHG DHGCollection DHG (mg) Concentration (mg) Concentration Rate (%)Concentration (wt. %) (wt. %) Rate (%) 32.7  0.11 6.89 0.17 21.1 1.4 GLYGLY Content GLY GLY Content GLY GLY Collection GLY (mg) Concentration(mg) Concentration Rate (%) Concentration (wt. %) (wt. %) Rate (%)62.70  0.21 32.64  0.80 52.1 3.8 DHG DHG Content DHG DHG Content DHG DHGCollection DHG (mg) Concentration (mg) Concentration Rate (%)Concentration (wt. %) (wt. %) Rate (%) 0.9  0.00 0.00 0.00  0.00  0.00

EQL represents equol; DZN represents daidzein; DHD representsdihydrodaidzin; GNT represents genistein; DHG representsdihydrogenistin; GLY represents glystein; and DHG representsdihydroglystein.

The isoflavone content (mg) denotes the gross weight of each isoflavonecontained in the EQL-containing fermented soybean hypocotyl (material:30 g) or in the obtained isolated extract (4100 mg); and the isoflavoneconcentration (wt. %) denotes the proportion of each isoflavonecontained in the EQL-containing fermented soybean hypocotyl (material)or in the obtained isolated extract. The “Concentration Rate” representsthe concentration rate of each component in the solid extract whenassuming the concentration of the component in the EQL-containingfermented soybean hypocotyl is 1.

The tables show that, by carrying out extraction on an equol-containingfermented soybean hypocotyl using an aqueous ethanol solution, the equolcollection rate increases and glystein was also collected. Meanwhile,the concentrations of other isoflavones were all very low in thisextract.

Example 1-3 Production of Equol-Containing Extract by Production MethodI

In this example, equol-containing fermented soybean hypocotyl powder wasproduced under the same conditions as in Reference Example 1-3 exceptthat a different lot of soybean hypocotyl powder was used.

50 g of the equol-containing fermented soybean hypocotyl powder wasmixed with 7 ml of purified water, and then further mixed with 250 ml ofn-hexane; and seven hours of stirring was performed at room temperature.Insoluble residue ware isolated by filtration, and seven hours of dryingwas performed at 60° C. to obtain defatted equol-containing fermentedsoybean hypocotyl powder.

20 g of the defatted equol-containing fermented soybean hypocotyl powderwas added to 100 ml of a 80 vol. % aqueous ethanol solution (100 mlconsisting of ethanol and 20 ml of water) or 100 ml of a 90 vol. %aqueous ethanol solution (100 ml consisting of ethanol and 10 ml ofwater), and fourteen hours of stirring was performed at roomtemperature. After removing the residue by filtration, the residue waswashed using 10 ml of an aqueous ethanol solution having the samecomposition as that of the extractant. The filtrate and the liquid afterwashing were isolated to obtain a liquid extract.

The liquid extract was dried for six hours at 60° C. to obtain a solidextract.

The weight of the obtained solid extract and the weight of equolcontained therein and the concentrations of daidzein, dihydrodaidzin,genistein, dihydrogenistin, glystein, dihydroglystein were measured, asshown in Tables 9 and 10.

TABLE 9 Extraction Results for 80 vol. % Ethanol Solution DefattedEQL-containing Fermented Soybean Hypocotyl powder Collected SolidExtract Amount for Dry Weight Weight Extraction (g) 20 (mg) 4000 EQL EQLContent EQL EQL Content EQL EQL Collection EQL (mg) Concentration (mg)Concentration Rate (%) Concentration (wt. %) (wt. %) Rate 107.60  0.54114.89  2.88 106.8  5.3 DZN DZN Content DZN DZN Content DZN DZNCollection DZN (mg) Concentration (mg) Concentration Rate (%)Concentration (wt. %) (wt. %) Rate (%) 4.00 0.02 0.00 0.00 0.0 0.0 DHDDHD Content DHD DHD Content DHD DHD Collection DHD (mg) Concentration(mg) Concentration Rate (%) Concentration (wt. %) (wt. %) Rate (%) 6.000.03 0.10 0.00 1.7 0.1 GNT GNT Content GNT GNT Content GNT GNTCollection GNT (mg) Concentration (mg) Concentration Rate (%)Concentration (wt. %) (wt. %) Rate (%) 4.60 0.02 0.28 0.01  0.01 0.4 DHGDHG Content DHG DHG Content DHG DHG Collection DHG (mg) Concentration(mg) Concentration Rate (%) Concentration (wt. %) (wt. %) Rate (%) 32.7 0.11 6.96 0.13 21.3  0.9 GLY GLY Content GLY GLY Content GLY GLYCollection GLY (mg) Concentration (mg) Concentration Rate (%)Concentration (wt. %) (wt. %) Rate (%) 41.80  0.21 24.03  0.60 57.5  2.9DHG DHG Content DHG DHG Content DHG DHG Collection DHG (mg)Concentration (mg) Concentration Rate (%) Concentration (wt. %) (wt. %)Rate (%) 0.6  0.00 0.00 0.00  0.00  0.00

EQL represents equol; DZN represents daidzein; DHD representsdihydrodaidzin; GNT represents genistein; DHG representsdihydrogenistin; GLY represents glystein; and DHG representsdihydroglystein.

The isoflavone content (mg) denotes the total weight of each isoflavonecontained in the defatted EQL-containing fermented soybean hypocotyl(material: 20 g) or in the collected extract (4000 mg); and theisoflavone concentration (wt. %) denotes the proportion of eachisoflavone contained in the defatted EQL-containing fermented soybeanhypocotyl (material) or in the collected extract. The “ConcentrationRate” represents the concentration rate of each component in the solidextract when assuming the concentration of the component in the defattedEQL-containing fermented soybean hypocotyl is 1.

TABLE 10 Extraction Results for 90 vol. % Ethanol Solution DefattedEQL-containing Fermented Soybean Hypocotyl powder Collected SolidExtract Amount for Dry Weight Weight Extraction (g) 20 (mg) 2200 EQL EQLContent EQL EQL Content EQL EQL Collection EQL (mg) Concentration (mg)Concentration Rate (%) Concentration (wt. %) (wt. %) Rate 107.60  0.54106.88  4.81 99.3 8.9 DZN DZN Content DZN DZN Content DZN DZN CollectionDZN (mg) Concentration (mg) Concentration Rate (%) Concentration (wt. %)(wt. %) Rate (%) 4.00 0.02 0.00 0.00  0.0 0.0 DHD DHD Content DHD DHDContent DHD DHD Collection DHD (mg) Concentration (mg) ConcentrationRate (%) Concentration (wt. %) (wt. %) Rate (%) 6.00 0.03 0.10 0.00  1.70.2 GNT GNT Content GNT GNT Content GNT GNT Collection GNT (mg)Concentration (mg) Concentration Rate (%) Concentration (wt. %) (wt. %)Rate (%) 4.60 0.02 0.21 0.01  0.01 0.4 DHG DHG Content DHG DHG ContentDHG DHG Collection DHG (mg) Concentration (mg) Concentration Rate (%)Concentration (wt. %) (wt. %) Rate (%) 32.7  0.11 6.96 0.21 21.3 1.5 GLYGLY Content GLY GLY Content GLY GLY Collection GLY (mg) Concentration(mg) Concentration Rate (%) Concentration (wt. %) (wt. %) Rate (%)41.80  0.21 21.97  0.99 52.6 4.7 DHG DHG Content DHG DHG Content DHG DHGCollection DHG (mg) Concentration (mg) Concentration Rate (%)Concentration (wt. %) (wt. %) Rate (%) 0.6  0.00 0.00 0.00  0.00  0.00

EQL represents equol; DZN represents daidzein; DHD representsdihydrodaidzin; GNT represents genistein; DHG representsdihydrogenistin; GLY represents glystein; and DHG representsdihydroglystein.

The isoflavone content (mg) denotes the gross weight of each isoflavonecontained in the defatted EQL-containing fermented soybean hypocotyl(material: 20 g) or in the collected extract (2200 mg); and theisoflavone concentration (wt. %) denotes the proportion of eachisoflavone contained in the defatted EQL-containing fermented soybeanhypocotyl (material) or in the collected extract. The “ConcentrationRate” represents the concentration rate of each component in the solidextract when assuming the concentration of the component in the defattedEQL-containing fermented soybean hypocotyl is 1.

The tables show that the high equol collection rate obtained in Example1-2 was ensured even when defatting the equol-containing fermentedsoybean hypocotyl in advance.

Example 1-4 Production of Equol-Containing Extract by Production MethodII

In this example, equol-containing fermented soybean hypocotyl powder(equol concentration=6.2 mg/g) was produced under the same conditions asin Reference Example 1-3 except that a different lot of soybeanhypocotyl powder was used.

80 g of the equol-containing fermented soybean hypocotyl powder wassuspended in 900 ml of water. 5 ml of acetic acid was added to thesuspension while it was being stirred, until the pH became approximately4.0. After 24 hours standing at room temperature, insoluble residue wascollected by filtration, and dried. 50.33 g of insoluble residue wasthus obtained.

Then, 250 ml of an aqueous ethanol solution (ethanol concentration=75vol. %) was added to the 50.33 g of insoluble residue, and two daysstirring was performed at room temperature. After removing the residueby filtration under reduced pressure, the filtrate was collected. Thefiltrate was evaporated to dryness into a solid with an evaporator toobtain 7.53 g of equol-containing powder.

The equol concentration of the obtained equol-containing powder was 60.0mg/g, which was about ten times the equol concentration (6.2 mg/g) ofthe equol-containing fermented soybean hypocotyl which was used as thestarting material. The recovery rate of equol was 91%.

Example 2 Equol Purification Example 2-1 Equol Purification Method 1

Equol-containing fermented soybean hypocotyl powder (equolconcentration=6.2 mg/g) was produced under the same conditions as inReference Example 1-3 except that a different lot of soybean hypocotylpowder was used.

10 g of the equol-containing fermented soybean hypocotyl powder wasadded to 50 ml of a 50 vol. % aqueous ethanol solution, and two hoursshaking was performed at room temperature. Thereafter, the residue(“Residue 1”, hereinafter) was removed by centrifugation (2500 rpm, 25minutes) to isolate a liquid extract (“Liquid Extract A-1”,hereinafter). Next, 50 ml of a 50 vol. % aqueous ethanol solution wasadded to Residue 1, and an hour of shaking was performed at roomtemperature. Thereafter, the residue was removed by centrifugation (2500rpm, 25 minutes) to isolate a liquid extract (“Liquid Extract A-2”,hereinafter). Extracts A-1 and A-2 were mixed and then evaporated todryness using an evaporator to obtain a residual solid (“Residual Solid1”, hereinafter).

Then, 10 ml of ethanol was added to Residual Solid 1, and 30 minutes ofshaking was performed at room temperature under supersonic treatment.Thereafter, the residue was removed by centrifugation (1000 rpm, 10minutes) to isolate a liquid extract (“Liquid Extract B”, hereinafter).Liquid Extract B was evaporated to dryness using an evaporator to obtainabout 1 g of a residual solid (“Residual Solid 2”, hereinafter).

Next, 10 ml of hexane was added to the 1 g of Residual Solid 2, and 30minutes of shaking was performed at room temperature. Then, the liquidextract was removed by centrifugation (1000 rpm, 10 minutes) to collect0.3 g of insoluble residue.

2 ml of a mixed solution of hexane and dimethylether(hexane:dimethylether=2:3 (volume ratio)) was added to the obtained 0.3mg of insoluble residue, and 30 minutes shaking was performed at roomtemperature. Then, the residue was removed by centrifugation (1000 rpm,10 minutes) to collect a liquid extract. The obtained liquid extract wassubjected to silica gel chromatography under the following conditions.

Column: A column 2 cm in internal diameter, filled with silica gel.

Mobile Phase:

200 ml of a mixed solution of hexane and diethylether(hexane:diethylether=40:60 (volume ratio));

200 ml of a mixed solution of hexane and diethylether(hexane:diethylether=30:70 (volume ratio));

200 ml of a mixed solution of hexane and diethylether(hexane:diethylether=25:75 (volume ratio));

200 ml of a mixed solution of hexane and diethylether(hexane:diethylether=20:80 (volume ratio));

and 200 ml of hexane were supplied sequentially.

Every 50 ml elution from the column was collected. The fraction elutedby the mixed solution of hexane and diethylether(hexane:diethylether=40:60 (volume ratio)) and the mixed solution ofhexane and diethylether (hexane:diethylether=25:75 (volume ratio))contained 40 mg of equol (recovery rate=80%), and the purity of equolwas 99.9% per dry solid. FIG. 8 shows the TLC Analysis of the fractioneluted using silica gel chromatography. As shown in the figure, thefraction eluted by the mixed solution of hexane and diethylether(hexane:diethylether=30:70 (volume ratio)) and the mixed solution ofhexane and diethylether (hexane:diethylether=25:75 (volume ratio))contained equol with high purity. Table 11 details the samples suppliedto the lanes of FIG. 8.

TABLE 11 Lane in FIG. 8 Sample Details Lane 1 Standard Product of EquolLane 2 Elution Fraction obtained while the elution amount of MixedSolution of Hexane and Diethylether (Hexane:Diethylether = 40:60(Capacity Ratio))ranges between 0 to 50 ml Lane 3 Elution Fractionobtained while the elution amount of Mixed Solution of Hexane andDiethylether (Hexane:Diethylether = 40:60 (Capacity Ratio))rangesbetween 50 to 100 ml Lane 4 Elution Fraction obtained while the elutionamount of Mixed Solution of Hexane and Diethylether (Hexane:Diethylether= 30:70 (Capacity Ratio))ranges between 0 to 50 ml Lane 5 ElutionFraction obtained while the elution amount of Mixed Solution of Hexaneand Diethylether (Hexane:Diethylether = 30:70 (Capacity Ratio))rangesbetween 50 to 100 ml Lane 6 Elution Fraction obtained while the elutionamount of Mixed Solution of Hexane and Diethylether (Hexane:Diethylether= 25:75 (Capacity Ratio))ranges between 0 to 50 ml Lane 7 ElutionFraction obtained while the elution amount of Mixed Solution of Hexaneand Diethylether (Hexane:Diethylether = 25:75 (Capacity Ratio))rangesbetween 50 to 100 ml Lane 8 Elution Fraction obtained while the elutionamount of Mixed Solution of Hexane and Diethylether (Hexane:Diethylether= 20:80 (Capacity Ratio))ranges between 0 to 50 ml

Example 2-2 Equal Purification Method 2

Equol-containing fermented soybean hypocotyl powder (equolconcentration=6.2 mg/g) was produced under the same conditions as inReference Example 1-3 except that a different lot of soybean hypocotylpowder was used.

100 g of the equol-containing fermented soybean hypocotyl powder wasadded to 500 ml of a 95 vol. % aqueous ethyl acetate solution, and fourhours of stirring was performed at room temperature. Thereafter,insoluble residue were removed by filtration to collect an extract. Theseparated insoluble residue was washed with 80 ml of ethyl acetate, andthe liquid after washing was mixed with the filtrate, and the mixturewas concentrated under reduced pressure to collect 12 g of oily matter.The oily matter was subjected to silica gel chromatography.

Column: A column 2 cm in internal diameter, filled with 70 g of silicagel using hexane.

Mobile phase: Supplied with 500 ml of hexane, and a mixed solution ofethyl acetate and hexane (ethyl acetate:hexane=1:5 (volume ratio))subsequently.

The solvent was distilled off from the equol-containing elution solventthus collected, and 630 mg of a yellow crystalline solid was obtained.

The crystalline solid was added to 2 ml of ethyl acetate and dissolvedby heating at 80° C. 8 ml of hexane was added thereto and 30 minutes ofstirring was performed while cooling the mixture with ice to precipitateequol crystals. The obtained equol crystals were collected byfiltration, and washed with hexane. Then the crystals were dried toobtain 530 mg of equol-containing solid (pale yellow crystals).

Further, the 530 mg equol-containing solid was added to 4 ml of aqueousethanol solution (ethanol concentration=62.5 vol. %), and heated to 80°C. to be dissolved in the solution. The solution was allowed to standfor twelve hours at 10° C. to recrystallize equol, and then filtered anddried. As a result, 390 mg of white needle-shaped equol crystals wereobtained.

The angle of rotation [α]_(D) ²⁵ of the obtained equol crystals was−21.6° in a 25° C. ethanol. The fusing point was 188-190° C. It wasconfirmed that the obtained equol was nearly 100% pure.

Example 3 Food Material Containing an Equol-containing Fermented SoybeanHypocotyl or Extract Thereof, and Foods Containing the Food MaterialExample 3-1 Production of Food Material Containing an Equal-containingFermented Soybean Hypocotyl and Cacao Mass

An equol-containing fermented soybean hypocotyl produced under the sameconditions as in Reference Examples 1-3 was mixed with cacao mass havingbeen dissolved by heat at a ratio of 1:3 (weight ratio). The mixture wascooled to obtain a food material in which an equol-containing fermentedsoybean hypocotyl is dispersed in cacao mass.

The taste of the food material thus produced was evaluated by 10panelists, with the result that all of the panelists concluded that thebitter taste derived from the equol-containing fermented soybeanhypocotyl was suppressed, and the food material possessed a pleasingflavor.

Example 3-2 Food Material Containing an Extract of Equol-ContainingFermented Soybean Hypocotyl

A solid extract (“75Et extract”, hereinafter) obtained by evaporatingLiquid Extract 2-1 produced in Example 1-1 using 75Et was mixed withcacao mass having been dissolved by heat at a ratio of 1:24 (weightratio). The mixture was cooled to obtain a food material in which a 75Etextract was dispersed in cacao mass.

The taste of the food material thus produced was evaluated by 10panelists, with the result that all of the panelists concluded that thebitter taste derived from the 75Et extract was suppressed, and the foodmaterial possessed a pleasing flavor.

Example 3-3 Baked Confectionery

The ingredients shown in Table 12 were mixed, and the mixture was formedinto a rectangle (about 1.5 cm×1.5 cm×10 cm) and baked in an oven at180° C. to produce a baked confectionery.

The taste of the baked confectionery thus produced was evaluated by 10panelists, with the result that all of the panelists concluded that thebitter taste derived from the equol-containing fermented soybeanhypocotyl was suppressed, and the baked confectionery possessed apleasing chocolate flavor and an original pleasing taste.

TABLE 12 Proportion (wt. %) Chip-shaped Food Material  5 Obtained inExample 3-1 (about 0.1 g per chip) Soy Flour 25 Raisins q.s. Butter q.s.Sugar q.s. Egg q.s. Orange Peel q.s. Pineapple q.s. Indigestible Dextrinq.s. Cacao Mass q.s. Sliced Almonds q.s. Cocoa Powder q.s. Salt q.s.Baking Powder q.s. Flavor q.s. Total 100 

Comparative Example 3-1 Baked Confectionery

A baked confectionery was produced under the same conditions as inExample 3-3 except that 1 g of the equol-containing fermented soybeanhypocotyl produced under the same conditions as in Example 1-3 was mixedwith 5 g of chocolate chips instead of the chip-shaped food materialobtained in Example 3-1.

The taste of the baked confectionery thus produced was evaluated by 10panelists, with the result that all of the panelists concluded that thepleasing taste disappeared and the bitter taste derived from theequol-containing fermented soybean hypocotyl remained throughout theconfectionery.

Example 3-4 Baked Confectionery

A baked confectionery was produced under the same conditions as inExample 3-3 except for the inclusion of the same amount of chip-shapedfood material obtained in Example 3-2 instead of the chip-shaped foodmaterials obtained in Example 3-1.

The taste of the baked confectionery thus produced was evaluated by 10panelists, with the result that all of the panelists concluded that thebitter taste derived from the 75Et extract was suppressed, and the bakedconfectionery possessed a pleasing chocolate flavor and an originalpleasing taste.

Example 4 Food Comprising Equol-Containing Fermented Soybean HypocotylMaterial or its Extract Example 4-A Beverage

Hereinafter, specific production examples of the beverage according tothe present invention are given.

Example 4-A-1 Sparkling Water

Sparkling water was obtained by preparing a mint-flavored syrup havingthe following composition, and mixing the resultant mint-flavored syrupwith carbonated water in a volumetric ratio of 3:1.

Composition of Mint-flavored Syrup Equol-containing fermented soybeanhypocotyl material 0.27 (Kg) (Reference Example 1-3) Sugar 0.60 Citricacid (crystalline) 0.01 Table Salt 0.012 Potassium chloride 0.005Calcium lactate 0.008 Peppermint extract 0.03 Peppermint flavor 0.20Water Balance Total 100.00 L

Example 4-A-2 Sparkling Water

Sparkling water was produced under the same conditions as in Example4-A-1, except that the mint-flavored syrup was prepared by adding,instead of the equol-containing fermented soybean hypocotyl material,0.034 kg of the solid extract prepared in Example 1-1 by evaporatingLiquid Extract 1-2 obtained using 75Et to dryness.

Example 4-A-3 Orange-Flavored Carbonated Beverage Containing No OrangeJuice

An orange-flavored carbonated beverage containing no orange juice wasobtained by preparing an orange-flavored syrup having the followingcomposition, and mixing the resultant orange-flavored syrup withcarbonated water in a volumetric ratio of 9:11.

Composition of Orange-flavored Syrup Equol-containing fermented soybeanhypocotyl material 0.20 (Kg) (Reference-Example 1-3) Sugar 10.00 Citricacid (crystalline) 0.03 DL-malic acid 0.07 Food coloring 0.05 Flavor0.10 Water Balance Total 45.00 L

Example 4-A-4 Orange-Flavored Carbonated Beverage Containing No OrangeJuice

An orange-flavored carbonated beverage containing no orange juice wasproduced under the same conditions as in Example 4-A-3, except that theorange-flavored syrup was prepared by adding, instead of theequol-containing fermented soybean hypocotyl material, 0.025 kg of thesolid extract prepared in Example 1-1 by evaporating Liquid Extract 1-2obtained using 75Et to dryness.

Example 4-A-5 Low-Calorie Cola

Sugarless cola was obtained by preparing a cola syrup having thefollowing composition, and mixing the resultant cola syrup withcarbonated water in a volumetric ratio of 8:12.

Composition of Cola Syrup Equol-containing fermented soybean hypocotylmaterial 0.20 (Kg) (Reference Example 1-2) Sucralose 0.02 Phosphoricacid 0.05 Citric acid (crystalline) 0.07 Caffeine 0.10 Caramel coloring0.20 Flavor (cola flavor) 0.10 Water Balance Total 40.00 L

Example 4-A-6 Low-Calorie Cola

Low-calorie cola was produced under the same conditions as in Example4-A-5, except that the cola syrup was prepared by adding, instead of theequol-containing fermented soybean hypocotyl material, 0.03 kg of thesolid extract prepared in Example 1-1 by evaporating Liquid Extract 1-2obtained using 75Et to dryness.

Example 4-A-7 Carbonated Apple Beverage

A carbonated apple beverage was obtained by preparing apple-flavoredsyrup having the following composition, and mixing the resultantapple-flavored syrup with carbonated water in a volumetric ratio of13:7.

Composition of Apple-flavored Syrup Equol-containing fermented soybeanhypocotyl material 0.20 (Kg) (Reference Example 1-2) 5 timesconcentrated apple juice 11.00 Fructose/glucose syrup 5.00 Citric acid(crystalline) 0.10 Food coloring 0.05 Flavor 0.10 Water Balance Total65.00 L

Example 4-A-8 Carbonated Apple Beverage

A carbonated apple beverage was produced under the same conditions as inExample 4-A-7, except that the apple-flavored syrup was prepared byadding, instead of the equol-containing fermented soybean hypocotylmaterial, 0.03 kg of the solid extract prepared in Example 1-1 byevaporating Liquid Extract 1-2 obtained using 75Et to dryness.

Example 4-A-9 Carbonated Grape Beverage

A carbonated grape beverage was obtained by preparing a grape-flavoredsyrup having the following composition, and mixing the resultantgrape-flavored syrup with carbonated water in a volumetric ratio of9:11.

Composition of Grape-flavored Syrup Equol-containing fermented soybeanhypocotyl material 0.20 (Kg) (Reference Example 1-1) 5 timesconcentrated clear grape juice 4.40 Citric acid (crystalline) 0.25 Foodcoloring 0.05 Flavor 0.20 Water Balance Total 45.00 L

Example 4-A-10 Carbonated Grape Beverage

A carbonated grape beverage was produced under the same conditions as inExample 4-A-9, except that the grape-flavored syrup was prepared byadding, instead of the equol-containing fermented soybean hypocotylmaterial, 0.03 kg of the solid extract prepared in Example 1-1 byevaporating Liquid Extract 1-2 obtained using 75Et to dryness.

Example 4-A-11 Beverage Containing Apple Juice

A beverage containing apple juice having the following composition wasproduced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-3) 5 times concentrated apple juice 22.00 Flavor0.05 Water Balance Total 100.00 L

Example 4-A-12 Beverage Containing Apple Juice

A beverage containing apple juice was produced under the same conditionsas in Example 4-A-11, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.05 kg of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-A-13 Beverage Containing Orange Juice

A beverage containing orange juice having the following composition wasproduced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-3) Concentrated Valencia orange juice (Brix: 55degree) 4.40 Sugar 5.00 Citric acid (crystalline) 0.16 Native gellan gum0.025 Vitamin C 0.03 Pectin 0.0025 Orange flavor 0.25 Water BalanceTotal 100.00 L

Example 4-A-14 Beverage Containing Orange Juice

A beverage containing orange juice was produced under the sameconditions as in Example 4-A-13, except for using, instead of theequol-containing fermented soybean hypocotyl material, 0.05 kg of thesolid extract prepared in Example 1-1 by evaporating Liquid Extract 1-2obtained using 75Et to dryness.

Example 4-A-15 Low-Calorie Beverage Containing Fruit Juice

A low-calorie beverage containing fruit juice having the followingcomposition was produced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-1) 5 times concentrated citrus juice 4.40 Sucralose0.009 Citric acid (crystalline) 0.18 L-ascorbic acid 0.03 Gellan gum0.024 Flavor 0.10 Water Balance Total 100.00 L

Example 4-A-16 Low-Calorie Beverage Containing Fruit Juice

A low-calorie beverage containing fruit juice was produced under thesame conditions as in Example 4-A-15, except for using, instead of theequol-containing fermented soybean hypocotyl material, 0.05 kg of thesolid extract prepared in Example 1-1 by evaporating Liquid Extract 1-2obtained using 75Et to dryness.

Example 4-A-17 Sugarless Near Water (Soft Drink Similar to Water)

Sugarless near water (soft drink similar to water) having the followingcomposition was produced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-3) Sucralose 0.008 Polydextrose 2.20 5 timesconcentrated grapefruit juice 0.44 Citric acid (crystalline) 0.53Calcium lactate 0.06 Potassium chloride 0.01 L-ascorbic acid 0.03Dibenzoyl thiamine hydrochloride 0.0002 Pyridoxine hydrochloride 0.00015Nicotinamide 0.0015 Flavor 0.10 Water Balance Total 100.00 L

Example 4-A-18 Sugarless Near Water (Soft Drink Similar to Water)

Sugarless near water (soft drink similar to water) was produced underthe same conditions as in Example 4-A-17, except for using, instead ofthe equol-containing fermented soybean hypocotyl material, 0.05 kg ofthe solid extract prepared in Example 1-1 by evaporating Liquid Extract1-2 obtained using 75Et to dryness.

Example 4-A-19 Sports Drink

A sports drink having the following composition was produced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-2) Clear lemon juice 0.50 Sucralose 0.02 Lacticminerals 1.00 Vitamin mix 0.25 Sodium L-ascorbate 0.05 Citric acid 0.125Water Balance Total 100.00 L

Example 4-A-20 Sports Drink

A sports drink was produced under the same conditions as in Example4-A-19, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 kg of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-A-21 Powdered Green Tea-milk Drink

A powdered green tea-milk drink having the following composition wasproduced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-2) Cow's milk 10.00 Skimmed milk 3.50 Powderedgreen tea 0.50 (RS-20, produced by ITO EN, LTD.) Sugar 7.00 Emulsifier0.70 Food coloring 0.03 Flavor 0.20 Sodium hydrogen carbonate foradjusting pH to 6.8 Water Balance Total 100.00 L

Example 4-A-22 Powdered Green Tea-milk Drink

A powdered green tea-milk drink was produced under the same conditionsas in Example 4-A-21, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.05 kg of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-A-23 Sugarless Yoghurt Drink

A sugarless yoghurt drink having the following composition was produced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-3) Fermented milk (SNF20%) 40.00 Water-solublesoybean polysaccharide 0.20 Pectin 0.20 Flavor 0.20 Lactic acid foradjusting pH to 4.2 Water Balance Total 100.00 L

Example 4-A-24 Sugarless Yoghurt Drink

A sugarless yoghurt drink was produced under the same conditions as inExample 4-A-23, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.05 kg of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-A-25 Pasteurized Lactic Acid Bacteria Beverage

A pasteurized lactic acid bacteria beverage having the followingcomposition was produced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-3) Fermented milk (SNF20%) 15.00 Water-solublesoybean polysaccharide 0.40 Sugar 8.00 Flavor 0.10 Lactic acid foradjusting pH to 3.8 Water Balance Total 100.00 L

Example 4-A-26 Pasteurized Lactic Acid Bacteria Beverage

A pasteurized lactic acid bacteria beverage was produced under the sameconditions as in Example 4-A-25, except for using, instead of theequol-containing fermented soybean hypocotyl material, 0.05 kg of thesolid extract prepared in Example 1-1 by evaporating Liquid Extract 1-2obtained using 75Et to dryness.

Example 4-A-27 Low-Calorie Sour Milk Beverage

A low-calorie sour milk beverage having the following composition wasproduced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-3) Skimmed milk 3.50 Water-soluble soybeanpolysaccharide 0.40 Sugar 5.00 Sucralose 0.0006 Fructose/glucose syrup(75%) 5.00 Citric acid (crystalline) 0.45 Flavor 0.10 Lactic acid foradjusting pH to 3.8 Water Balance Total 100.00 L

Example 4-A-28 Low-Calorie Sour Milk Beverage

Low-calorie sour milk beverage was produced under the same conditions asin Example 4-A-27, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.05 kg of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-A-29 Strawberry-Flavored Milk Drink

A strawberry-flavored milk drink having the following composition wasproduced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-3) Skimmed milk 2.80 Whole milk powder 3.00 Sugar12.00 Emulsifier 0.06 Food coloring 0.05 Flavor (strawberry flavor) 0.10Water Balance Total 100.00 L

Example 4-A-30 Strawberry-Flavored Milk Drink

Strawberry-flavored milk drink was produced under the same conditions asin Example 4-A-29, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.05 kg of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-A-31 Non-Fat Milk

Non-fat milk having the following composition was produced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-1) Skimmed milk 12.00 Egg calcium 1.00 Emulsifier0.06 Flavor 0.10 Water Balance Total 100.00 L

Example 4-A-32 Non-Fat Milk

Non-fat milk was produced under the same conditions as in Example4-A-31, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 kg of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-A-33 Cream Soda

A cream soda having the following composition was produced.

Equol-containing fermented soybean hypocotyl material 0.20 (Kg)(Reference Example 1-1) Skimmed sweetened condensed milk 3.00 Citricacid (crystalline) 0.04 Sugar 15.00 Food coloring 0.60 Flavor 0.10 WaterBalance Total 40.00 L

Example 4-A-34 Cream Soda

A cream soda was produced under the same conditions as in Example4-A-33, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.025 kg of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-A-35 Powdered Green Tea-Milk Drink

A powdered green tea-milk drink having the following composition wasproduced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-2) Skimmed milk 3.50 Cow's milk 10.00 Emulsifier0.70 Sugar 6.00 Water Balance Total 100.00 L

Example 4-A-36 Powdered Green Tea-Milk Drink

Powdered green tea-milk drink was produced under the same conditions asin Example 4-A-35, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.05 kg of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-A-37 Black Tea

Black tea having the following composition was produced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-2) Darjeeling tea extract (40 times) 20.00 Sugar5.00 Flavor 0.05 Water Balance Total 100.00 L

Example 4-A-38 Black Tea

Black tea was produced under the same conditions as in Example 4-A-37,except for using, instead of the equol-containing fermented soybeanhypocotyl material, 0.05 kg of the solid extract prepared in Example 1-1by evaporating Liquid Extract 1-2 obtained using 75Et to dryness.

Example 4-A-39 Beverage Containing Apple Juice

A beverage containing apple juice having the following composition wasproduced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-3) 5 times concentrated clear apple juice 2.20Oligosaccharide 1.00 Reducing maltose syrup 1.50 Vitamin C 0.05 Citricacid 0.10 Water-soluble dietary fiber 8.00 Water Balance Total 100.00 L

Example 4-A-40 Beverage Containing Apple Juice

A beverage containing apple juice was produced under the same conditionsas in Example 4-A-39, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.05 kg of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-A-41 Soft Drink

A soft drink having the following composition was produced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-3) Fructose 3.70 Fructose/glucose syrup 8.00 Sugar0.30 DL-malic acid 0.07 Sodium citrate 0.03 Emulsifier 0.052 Flavor 0.22Water Balance Total 100.00 L

Example 4-A-42 Soft Drink

A soft drink was produced under the same conditions as in Example4-A-41, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 kg of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-A-43 Beverage Containing Orange Juice

A beverage containing orange juice having the following composition wasproduced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-3) Fructose/glucose syrup 2.50 Beet oligosaccharide0.026 Sugar 4.00 Concentrated orange juice 4.40 Citric acid 0.026 Sodiumcitrate 0.003 L-ascorbic acid 0.01 Flavor 0.10 Water Balance Total100.00 L

Example 4-A-44 Beverage Containing Orange Juice

A beverage containing orange juice was produced under the sameconditions as in Example 4-A-43, except for using, instead of theequol-containing fermented soybean hypocotyl material, 0.05 kg of thesolid extract prepared in Example 1-1 by evaporating Liquid Extract 1-2obtained using 75Et to dryness.

Example 4-A-45 Beverage Containing Apple Juice

A beverage containing apple juice having the following composition wasproduced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-3) Concentrated apple juice 4.40 Sugar 8.00 Citricacid 0.15 Flavor 0.12 Water Balance Total 100.00 L

Example 4-A-46 Beverage Containing Apple Juice

A beverage containing apple juice was produced under the same conditionsas in Example 4-A-45, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.05 kg of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-A-47-1 Vegetable Juice

Vegetable juice having the following composition was produced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-2) Vegetable fruit juice 20.00 Water Balance Total100.00 L

Example 4-A-47-2 Vegetable Juice

Vegetable juice was produced under the same conditions as in Example4-A-47-1, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 kg of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-A-48-1 Sports Drink

A sports drink having the following composition was produced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-2) Lactic minerals 1.00 Vitamin mix 0.25 Citricacid 0.125 Sodium L-ascorbate 0.05 Lemon juice 0.50 Sugar 12.00 WaterBalance Total 100.00 L

Example 4-A-48-2 Sports Drink

A sports drink was produced under the same conditions as in Example4-A-48-1, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 kg of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-A-49 Oolong Tea

Oolong tea having the following composition was produced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-3) Oolong tea extract 3.00 Sodium L-ascorbate 0.01Flavor 0.20 Water Balance Total 100.00 L

Example 4-A-50 Oolong Tea

Oolong tea was produced under the same conditions as in Example 4-A-49,except for using, instead of the equol-containing fermented soybeanhypocotyl material, 0.05 kg of the solid extract prepared in Example 1-1by evaporating Liquid Extract 1-2 obtained using 75Et to dryness.

Example 4-A-51 Tea with Lemon

Tea with lemon having the following composition was produced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-3) Black tea extract 6.00 Reducing maltose syruppowder 3.00 Sugar 3.00 Lemon juice 0.50 L-ascorbic acid 0.02 Flavor 0.10Water Balance Total 100.00 L

Example 4-A-52 Tea with Lemon

Tea with lemon was produced under the same conditions as in Example4-A-51, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 kg of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-A-53 Tea with Milk

Tea with milk having the following composition was produced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-3) Cow's milk 7.00 Whole milk powder 0.25 Sugar4.00 Tea extract 35.00 Emulsifier 0.05 Flavor 0.10 Water Balance Total100.00 L

Example 4-A-54 Tea with Milk

Tea with milk was produced under the same conditions as in Example4-A-53, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 kg of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-A-55 Coffee Drink

A coffee drink having the following composition was produced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-1) Coffee extract 30.00 Cow's milk 25.00 Emulsifier0.10 Sodium bicarbonate 0.12 Stevia 0.01 Sugar 3.00 L-rhamnose 0.01Flavor 0.05 Water Balance Total 100.00 L

Example 4-A-56 Coffee Drink

A coffee drink was produced under the same conditions as in Example4-A-55, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 kg of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-A-57 Cocoa Milk Drink

A Cocoa milk drink having the following composition was produced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-1) Cow's milk 10.00 Skimmed milk 3.50 Fructose 5.50Sugar 0.40 Cocoa powder 1.00 Emulsifier 0.45 Flavor 0.08 Water BalanceTotal 100.00 L

Example 4-A-58 Cocoa Milk Drink

A cocoa milk drink was produced under the same conditions as in Example4-A-57, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 kg of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-A-59 Powdered Green Tea-Milk Drink

A powdered green tea-milk drink having the following composition wasproduced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-1) Cow's milk 10.00 Skimmed milk 3.50 Fructose 5.80Sugar 0.40 Powdered green tea 0.90 Emulsifier 0.48 Flavor 0.27 Foodcoloring 0.03 Water Balance Total 100.00 L

Example 4-A-60 Powdered Green Tea-Milk Drink

A powdered green tea-milk drink was produced under the same conditionsas in Example 4-A-59, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.05 kg of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-A-61 Sour Milk Beverage

A sour milk beverage having the following composition was produced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-3) Skimmed milk 1.50 Fructose 2.50 Sugar 0.50Concentrated strawberry juice 0.22 Sodium citrate 0.32 Emulsifier 0.40Flavor 0.12 Water Balance Total 100.00 L

Example 4-A-62 Sour Milk Beverage

A sour milk beverage was produced under the same conditions as inExample 4-A-61, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.05 kg of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-A-63 Beverage Containing Vitamins

A beverage containing vitamins having the following composition wasproduced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-3) Sugar 24.00 Citric acid 0.20 L-ascorbic acid0.30 Vitamin mix 0.06 Flavor 0.10 Carbonated water Balance Total 100.00L

Example 4-A-64 Beverage Containing Vitamins

A beverage containing vitamins was produced under the same conditions asin Example 4-A-63, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.05 kg of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-A-65 Red Bean Soup

A red bean soup of the following composition was produced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-3) Red bean paste 10.00 Sugar alcohol 6.50 Sugar24.00 Table salt 0.10 Emulsifier 0.60 Flavor 0.10 Water Balance Total100.00 L

Example 4-A-66 Red Bean Soup

A red bean soup was produced under the same conditions as in Example4-A-65, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 kg of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-A-67 Green Tea

Green tea having the following composition was produced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-1) Green tea extract 3.00 Sodium L-ascorbate 0.01Flavor 0.20 Water Balance Total 100.00 L

Example 4-A-68 Green Tea

Green tea was produced under the same conditions as in Example 4-A-67,except for using, instead of the equol-containing fermented soybeanhypocotyl material, 0.05 kg of the solid extract prepared in Example 1-1by evaporating Liquid Extract 1-2 obtained using 75Et to dryness.

Example 4-A-69 Powdered Orange Juice Drink

A powdered orange juice drink having the following composition wasproduced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-2) Fructose/glucose syrup 2.50 Beet oligosaccharide0.026 Sugar 4.00 Powdered orange juice 2.00 Citric acid 0.026 Sodiumcitrate 0.003 L-ascorbic acid 0.01 Flavor 0.10 Water Balance Total100.00 L

Example 4-A-70 Powdered Orange Juice Drink

A powdered orange juice drink was produced under the same conditions asin Example 4-A-69, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.05 kg of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-A-71 Amino Acid Drink

An amino acid drink having the following composition was produced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-3) Sugar 15.00 Fructose 15.00 Citric acid 4.00Sodium citrate 1.00 L-valine 2.00 L-leucine 4.00 L-isoleucine 2.00L-arginine 2.00 Flavor 0.10 Water Balance Total 100.00 L

Example 4-A-72 Amino Acid Drink

An amino acid drink was produced under the same conditions as in Example4-A-71, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 kg of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-A-73 Soya Milk Drink

A soya milk drink having the following composition was produced.

Equol-containing fermented soybean 0.40 (part by weight) hypocotylmaterial (Reference Example 1-3) Soya Milk 100.00

Example 4-A-74 Soya Milk Drink

A soya milk drink was produced under the same conditions as in Example4-A-73, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 parts by weight of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-A-75 Energy Drink

An energy drink having the following composition was produced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-3) Taurine 1.00 Nicotinamide 0.02 Vitamin B1 0.005Vitamin B2 0.005 Vitamin B6 0.005 Caffeine 0.05 Sugar 15.00 Fructose15.00 Citric acid 0.10 Flavor 0.10 Water Balance Total 100.00 L

Example 4-A-76 Amino Acid Drink

An amino acid drink was produced under the same conditions as in Example4-A-75, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 kg of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-B Dietary Supplement

Hereinafter, specific production examples of the dietary supplementaccording to the present invention are given.

Example 4-B-1 Softgel

A softgel was produced according to a known method usingequol-containing fermented soybean hypocotyl material (Reference Example1-3), gelatin, glycerine, citric acid, soybean oil, beeswax, lecithin, βcarotene, and water.

Example 4-B-2 Softgel

A softgel was produced under the same conditions as in Example 4-B-1,except for using, instead of the equol-containing fermented soybeanhypocotyl material, the solid extract prepared in Example 1-1 byevaporating Liquid Extract 1-2 obtained using 75Et to dryness.

Example 4-C Creamy Food

Hereinafter, specific production examples of the creamy food accordingto the present invention are given.

Example 4-C-1 Whipped Cream

Whipped cream having the following composition was produced.

Equol-containing fermented soybean hypocotyl material 0.40 (g)(Reference Example 1-3) Coconut oil (mp: 35° C.) 27.00 Glycerine fattyacid ester 0.10 Skimmed milk 3.30 Sucralose 0.012 Reducing starch sugar25.00 Lecithin 0.15 Emulsifier 0.70 Sodium metaphosphate 0.10 WaterBalance Total 100.00 g

Example 4-C-2 Whipped Cream

Whipped cream was produced under the same conditions as in Example4-C-1, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 g of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-C-3 Coffee Whitener

A coffee whitener having the following composition was produced.

Equol-containing fermented soybean hypocotyl material 0.40 (g)(Reference Example 1-3) Purified coconut oil 36.00 Sodium casein 4.50Skimmed milk 5.00 Lecithin 0.05 Trisodium citrate 0.20 Sucrose fattyacid ester 0.40 Milk flavor 0.10 Water Balance Total 100.00 g

Example 4-C-4 Coffee Whitener

A coffee whitener was produced under the same conditions as in Example4-C-3, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 g of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-C-5 Custard

Custard having the following composition was produced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-3) Margarine 20.00 Starch syrup 5.00 Whole egg 3.00Cornstarch 4.00 Modified starch 6.00 Lactic protein 2.00 Sugar 30.00Water Balance Total 100.00 kg

Example 4-C-6 Custard

Custard was produced under the same conditions as in Example 4-C-5,except for using, instead of the equol-containing fermented soybeanhypocotyl material, 0.05 kg of the solid extract prepared in Example 1-1by evaporating Liquid Extract 1-2 obtained using 75Et to dryness.

Example 4-C-7 Custard

Custard having the following composition was produced.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-3) Carrageenan 0.40 Gelatin 0.50 Cornstarch 4.00Corn syrup solids 3.00 Granulated sugar 10.00 Glucose 7.00 Skimmed milk4.00 Whole egg 3.50 Isomerized sugar 7.00 Skimmed sweetened condensedmilk 3.00 Unsalted butter 6.50 Water 50.00

Example 4-C-8 Custard

Custard was produced under the same conditions as in Example 4-C-7,except for using, instead of the equol-containing fermented soybeanhypocotyl material, 0.05 kg of the solid extract prepared in Example 1-1by evaporating Liquid Extract 1-2 obtained using 75Et to dryness.

Example 4-D Dessert

Hereinafter, specific production examples of the dessert according tothe present invention are given.

Example 4-D-1 Hard Yoghurt

The ingredients of the following composition were homogenized, and astarter yoghurt was added thereto to be fermented, thereby producinghard yoghurt.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-3) Skimmed milk 9.00 Unsalted butter 5.00 Sugar6.50 Gelling agent 0.60 Water Balance Total 100.00 kg

Example 4-D-2 Hard Yoghurt

Hard yoghurt was produced under the same conditions as in Example 4-D-1,except for using, instead of the equol-containing fermented soybeanhypocotyl material, 0.05 kg of the solid extract prepared in Example 1-1by evaporating Liquid Extract 1-2 obtained using 75Et to dryness.

Example 4-D-3 Soft Yoghurt

The ingredients of the following composition were homogenized, and astarter yoghurt was added thereto to be fermented, thereby producingsoft yoghurt.

Equol-containing fermented soybean hypocotyl material 0.40 (Kg)(Reference Example 1-3) Skimmed milk 9.00 Butter 5.25 Sugar 6.50 Locustbean gum 0.30 Water Balance Total 100.00 kg

Example 4-D-4 Soft Yoghurt

Soft yoghurt was produced under the same conditions as in Example 4-D-3,except for using, instead of the equol-containing fermented soybeanhypocotyl material, 0.05 kg of the solid extract prepared in Example 1-1by evaporating Liquid Extract 1-2 obtained using 75Et to dryness.

Example 4-D-5 White Peach Jelly

A white peach jelly was produced in accordance with a general productionmethod of a jelly, using the ingredients of the following composition.

Equol-containing fermented soybean 0.40 (part by weight) hypocotylmaterial (Reference Example 1-3) Sugar 14.40 κ-Carrageenan 0.60 Whitepeach puree 20.00 Water Balance Total 100.00 parts by weight

Example 4-D-6 White Peach Jelly

A white peach jelly was produced under the same conditions as in Example4-D-5, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 parts by weight of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-D-7 Grapefruit Jelly

A grapefruit jelly was produced in accordance with a general productionmethod of a jelly, using the ingredients of the following composition.

Equol-containing fermented soybean 0.40 (part by weight) hypocotylmaterial (Reference Example 1-2) Sugar 18.00 Gellan gum 0.25 Grapefruitjuice 20.00 Calcium lactate 0.10 Citric acid 0.20 Flavor 0.10 WaterBalance Total 100.00 parts by weight

Example 4-D-8 Grapefruit Jelly

A grapefruit jelly was produced under the same conditions as in Example4-D-7, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 parts by weight of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-D-9 Coffee Jelly

A coffee jelly was produced in accordance with a general productionmethod of a jelly, using the ingredients of the following composition.

Equol-containing fermented soybean 0.40 (part by weight) hypocotylmaterial (Reference Example 1-2) Reducing starch syrup 10.00 Xylitol5.00 κ-Carrageenan 0.50 Agar 0.20 Locust bean gum 0.20 Sugar 3.00 Flavor0.15 Water Balance Total 100.00 parts by weight

Example 4-D-10 Coffee Jelly

A coffee jelly was produced under the same conditions as in Example4-D-9, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 parts by weight of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-D-11 Strawberry Milk Jelly

A strawberry milk jelly was produced in accordance with a generalproduction method of a jelly, using the ingredients of the followingcomposition.

Equol-containing fermented soybean 0.40 (part by weight) hypocotylmaterial (Reference Example 1-2) Purified coconut oil 30.00 Emulsifier0.25 Skimmed milk 3.30 Sugar 3.50 Reducing starch syrup 20.00 Lactitol8.00 Stabilizer 0.70 Xylitol 5.00 Gelatin 0.90 Strawberry puree 5.00Concentrated strawberry juice 5.00 Citric acid 0.10 Water Balance Total100.00 parts by weight

Example 4-D-12 Strawberry Milk Jelly

A strawberry milk jelly was produced under the same conditions as inExample 4-D-11, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.05 parts by weight of the solidextract prepared in Example 1-1 by evaporating Liquid Extract 1-2obtained using 75Et to dryness.

Example 4-E Snack

Hereinafter, specific production examples of the snack according to thepresent invention are given.

Example 4-E-1 Hard Candy

A hard candy was produced in accordance with a general production methodof a hard candy, using the ingredients of the following composition.

Equol-containing fermented soybean hypocotyl material 4.00 (g)(Reference Example 1-3) Sugar 70.00 Starch syrup 40.00 Water 20.00

Example 4-E-2 Hard Candy

A hard candy was produced under the same conditions as in Example 4-E-1,except for using, instead of the equol-containing fermented soybeanhypocotyl material, 0.5 g of the solid extract prepared in Example 1-1by evaporating Liquid Extract 1-2 obtained using 75Et to dryness.

Example 4-E-3 Ginger Candy

A ginger candy was produced in accordance with a general productionmethod of a candy, using the ingredients of the following composition.

Equol-containing fermented soybean hypocotyl material 4.00 (g)(Reference Example 1-1) Ginger paste 1.50 Reducing lactose 60.00Reducing maltose syrup 56.00 Sucralose 0.03 Flavor 0.075 Water 30.00

Example 4-E-4 Ginger Candy

A ginger candy was produced under the same conditions as in Example4-E-3, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.5 g of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-E-5 Throat Soothing Herb Candy

A throat soothing herb candy was produced in accordance with a generalproduction method of a candy, using the ingredients of the followingcomposition.

Equol-containing fermented soybean hypocotyl material 4.00 (g)(Reference Example 1-2) Mixed herb extract 0.60 Sugar 40.00 Reducingmaltose syrup 56.00 Peppermint Flavor 0.20 Caramel coloring 0.20 Water20.00

Example 4-E-6 Throat Soothing Herb Candy

A throat soothing herb candy was produced under the same conditions asin Example 4-E-5, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.5 g of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-E-7 Lemon Gummy Candy

A lemon gummy candy was produced in accordance with a general productionmethod of a gummy candy, using the ingredients of the followingcomposition.

Equol-containing fermented soybean hypocotyl material 4.00 (g)(Reference Example 1-2) Acid gelatin (250 blooms) 7.00 Reducing starchsyrup 100.00 4 times concentrated lemon juice 1.25 Citric acid(crystalline) 1.00 Sucralose 0.02 Food coloring 0.02 Flavor 0.02 Water12.00

Example 4-E-8 Lemon Gummy Candy

A lemon gummy candy was produced under the same conditions as in Example4-E-7, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.5 g of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-E-9 Caramel Candy

A caramel candy was produced in accordance with a general productionmethod of a caramel candy, using the ingredients of the followingcomposition.

Equol-containing fermented soybean hypocotyl material 4.00 (g)(Reference Example 1-1) Sweetened condensed milk 45.00 Starch syrup45.00 Vegetable fat and oil 3.50 Lecithin 0.20 Fondant 5.00 Flavor(caramel flavor) 0.10

Example 4-E-10 Caramel Candy

A caramel candy was produced under the same conditions as in Example4-E-9, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.5 g of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-E-11 Pretzel

The following ingredients were mixed to prepare dough, which was bakedin an oven; thereby, a pretzel was produced.

Equol-containing fermented soybean hypocotyl material 4.00 (g)(Reference Example 1-1) Strong flour 100.00 Baking powder 1.35 Unsaltedbutter 25.00 Table salt 2.00 Sugar 17.50 Soybean dietary fiber 0.80Sucrose fatty acid ester 0.30 Flavor 0.20 Water 40.00

Example 4-E-12 Pretzel

A pretzel was produced under the same conditions as in Example 4-E-11,except for using, instead of the equol-containing fermented soybeanhypocotyl material, 0.5 g of the solid extract prepared in Example 1-1by evaporating Liquid Extract 1-2 obtained using 75Et to dryness.

Example 4-E-13 Mint Tablet

A mint tablet was produced in accordance with a general productionmethod of a tablet, using the ingredients of the following composition.

Equol-containing fermented soybean hypocotyl material 4.00 (g)(Reference Example 1-3) Sorbitol 90.00 Sucralose 0.15 Peppermint flavor5.00 Sucrose fatty acid ester 1.00

Example 4-E-14 Mint Tablet

A mint tablet was produced under the same conditions as in Example4-E-13, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.5 g of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-E-15 Yoghurt Tablet

A yoghurt tablet was produced in accordance with a general productionmethod of a tablet, using the ingredients of the following composition.

Equol-containing fermented soybean hypocotyl material 4.00 (g)(Reference Example 1-2) Trehalose (granulation) 88.60 Sucralose 0.08Powdered fermented milk 5.00 Anhydrous citric acid 1.00 Yogurt flavor0.20 Sucrose fatty acid ester 1.50

Example 4-E-16 Yoghurt Tablet

A yoghurt tablet was produced under the same conditions as in Example4-E-15, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.5 g of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-E-17 Lemon Tablet

A lemon tablet was produced in accordance with a general productionmethod of a tablet, using the ingredients of the following composition.

Equol-containing fermented soybean hypocotyl material 4.00 (g)(Reference Example 1-1) Sugar (granulation) 89.50 Powdered lemon juice2.00 L-ascorbic acid 3.00 Anhydrous citric acid 1.00 Sucrose fatty acidester 1.00

Example 4-E-18 Lemon Tablet

A lemon tablet was produced under the same conditions as in Example4-E-17, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.5 g of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-E-19 Mint Chewing Gum

Mint chewing gum was produced in accordance with a general productionmethod of a chewing gum, using the ingredients of the followingcomposition.

Equol-containing fermented soybean hypocotyl material 4.00 (g)(Reference Example 1-1) Gum base 25.00 Lactitol 63.20 Sugar alcohol 5.00Sucralose 0.13 Glycerine 0.50 Peppermint oil 1.50 Mint Flavor 0.50 Foodcoloring 0.20

Example 4-E-20 Mint Chewing Gum

Mint chewing gum was produced under the same conditions as in Example4-E-19, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.5 g of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-E-21 Apple Bubble Gum

Apple bubble gum was produced in accordance with a general productionmethod of a chewing gum, using the ingredients of the followingcomposition.

Equol-containing fermented soybean hypocotyl material 4.00 (g)(Reference Example 1-1) Gum base 30.00 Palatinit 70.00 Sucralose 0.084Glycerine 0.50 Citric acid (crystalline) 1.00 Apple flavor 0.80

Example 4-E-22 Apple Bubble Gum

Apple bubble gum was produced under the same conditions as in Example4-E-21, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.5 g of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-E-23 Strawberry Chewing Gum

Strawberry chewing gum was produced in accordance with a generalproduction method of a chewing gum, using the ingredients of thefollowing composition.

Equol-containing fermented soybean hypocotyl material 4.00 (g)(Reference Example 1-3) Gum base 23.00 Powdered sugar 62.00 Starch syrup5.00 Sucralose 0.001 Citric acid (crystalline) 1.20 Red cabbage color0.20 Strawberry flavor 0.20

Example 4-E-24 Strawberry Chewing Gum

Strawberry chewing gum was produced under the same conditions as inExample 4-E-23, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.5 g of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-E-25 Chocolate

Chocolate was produced in accordance with a general production method ofchocolate, using the ingredients of the following composition.

Equol-containing fermented soybean hypocotyl material 4.00 (g)(Reference Example 1-3) Cacao mass 40.00 Cocoa butter 25.00 Reducedpowder sugar 50.00 Sucralose 0.03 Lecithin 0.40 Vanillin 0.05

Example 4-E-26 Chocolate

Chocolate was produced under the same conditions as in Example 4-E-25,except for using, instead of the equol-containing fermented soybeanhypocotyl material, 0.5 g of the solid extract prepared in Example 1-1by evaporating Liquid Extract 1-2 obtained using 75Et to dryness.

Example 4-E-27 Strawberry-flavored Chocolate

Strawberry-flavored chocolate was produced in accordance with a generalproduction method of chocolate, using the ingredients of the followingcomposition.

Equol-containing fermented 4.00 (g) soybean hypocotyl material(Reference Example 1-3) Cocoa butter 25.00 Hard butter 7.00 Whole milkpowder 27.00 Sugar 41.00 Strawberry flavor 0.20 Beet red 0.50 Sucralose0.001 Lecithin 0.30 Vanillin 0.02

Example 4-E-28 Strawberry-Flavored Chocolate

Strawberry-flavored chocolate was produced under the same conditions asin Example 4-E-27, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.5 g of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-E-29 Cookie

The following ingredients were mixed to prepare dough, which was bakedin an oven to produce a cookie.

Equol-containing fermented 0.40 (part by weight) soybean hypocotylmaterial (Reference Example 1-3) Soft flour 48.50 Baking powder 0.30Unsalted margarine 24.00 Table salt 0.20 Sugar 12.00 Egg yolk 4.70Trehalose 22.00 Flavor 0.20 Water 40.00

Example 4-E-30 Cookie

A cookie was produced under the same conditions as in Example 4-E-29,except for using, instead of the equol-containing fermented soybeanhypocotyl material, 0.05 parts by weight of the solid extract preparedin Example 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-E-31 Skin of Manju (a Bun Filled with Steamed Azuki Bean Jam)

The skin of a manju (a bun filled with steamed azuki bean jam) wasproduced in accordance with a general production method of a skin ofmanju, using the ingredients of the following composition.

Equol-containing fermented 4.00 (part by weight) soybean hypocotylmaterial (Reference Example 1-2) Soft flour 500.00 Baking powder 2.50Xanthan gum 1.00 Table salt 6.00 Sugar 30.00 Trehalose 30.00 Dry yeast7.50 Lard 15.00 Water 200.00

Example 4-E-32 Skin of Manju (a Bun Filled with Steamed Azuki Bean Jam)

The skin of a manju (a bun filled with steamed azuki bean jam) wasproduced under the same conditions as in Example 4-E-31, except forusing, instead of the equol-containing fermented soybean hypocotylmaterial, 0.5 parts by weight of the solid extract prepared in Example1-1 by evaporating Liquid Extract 1-2 obtained using 75Et to dryness.

Example 4-E-33 Apple Pie Filling

An apple pie filling was produced using the following ingredients.

Equol-containing fermented 0.40 (part by weight) soybean hypocotylmaterial (Reference Example 1-1) Apple 200.00 Maltose 150.00 Sucralose0.10 Lemon juice 10.00 Walnut 60.00 Raisin 60.00 Apple flavor 0.20

Example 4-E-34 Apple Pie Filling

An apple pie filling was produced under the same conditions as inExample 4-E-33, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.05 parts by weight of the solidextract prepared in Example 1-1 by evaporating Liquid Extract 1-2obtained using 75Et to dryness.

Example 4-E-35 Pound Cake

The following ingredients were mixed to prepare dough, which was bakedin an oven to produce a pound cake.

Equol-containing fermented 0.40 (part by weight) soybean hypocotylmaterial (Reference Example 1-1) Butter 100.00 Reducing maltose syrup100.00 Sucralose 0.03 Stevia 0.03 Whole egg 100.00 Flavor 0.22 Softflour 120.00 Baking powder 1.50

Example 4-E-36 Pound Cake

A pound cake was produced under the same conditions as in Example4-E-35, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 parts by weight of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-E-37 Bracken-Starch Dumpling

A bracken-starch dumpling was produced using the following ingredients.

Equol-containing fermented 0.40 (part by weight) soybean hypocotylmaterial (Reference Example 1-1) Rice starch 20.00 Sweet potato starch40.00 Sugar 6.00 Water Balance Total 100.00 parts by weight

Example 4-E-38 Bracken-Starch Dumpling

A bracken-starch dumpling was produced under the same conditions as inExample 4-E-37, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.05 parts by weight of the solidextract prepared in Example 1-1 by evaporating Liquid Extract 1-2obtained using 75Et to dryness.

Example 4-E-39 Bean Confectionery

A bean confectionery was produced using the following ingredients.

Equol-containing fermented 6.00 (part by weight) soybean hypocotylmaterial (Reference Example 1-3) Kanbaiko 7.00 (ground toasted ricecake) Sugar 53.00 Kinako (roasted soybean powder) 4.00 Baking powderq.s. Flavor q.s. Sweetener q.s. Total 100.00 parts by weight

Example 4-E-40 Bean Confectionery

A bean confectionery was produced under the same conditions as inExample 4-E-39, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.75 parts by weight of the solidextract prepared in Example 1-1 by evaporating Liquid Extract 1-2obtained using 75Et to dryness.

Example 4-E-41 Bean Confectionery

A bean confectionery was produced using the following ingredients.

Equol-containing fermented 12.00 (part by weight) soybean hypocotylmaterial (Reference Example 1-2) Rice flour 13.00 Sugar 45.00 Kinako(roasted soybean powder) 15.00 Powdered green tea 15.00 Baking powderq.s. Flavor q.s. Sweetener q.s. Total 100.00 parts by weight

Example 4-E-42 Bean Confectionery

A bean confectionery was produced under the same conditions as inExample 4-E-41, except for using, instead of the equol-containingfermented soybean hypocotyl material, 1.5 parts by weight of the solidextract prepared in Example 1-1 by evaporating Liquid Extract 1-2obtained using 75Et to dryness.

Example 4-E-43 Bean Confectionery

A bean confectionery was produced using the following ingredients.

Equol-containing fermented 18.00 (part by weight) soybean hypocotylmaterial (Reference Example 1-1) Kanbaiko 15.00 (ground toasted ricecake) Sugar 52.00 Kinako (roasted soybean powder) 15.00 Powdered greentea 12.00 Baking powder q.s. Flavor q.s. Sweetener q.s. Total 100.00parts by weight

Example 4-E-44 Bean Confectionery

A bean confectionery was produced under the same conditions as inExample 4-E-43, except for using, instead of the equol-containingfermented soybean hypocotyl material, 2.25 parts by weight of the solidextract prepared in Example 1-1 by evaporating Liquid Extract 1-2obtained using 75Et to dryness.

Example 4-E-45 Bean Confectionery

A bean confectionery was produced using the following ingredients.

Equol-containing fermented 6.00 (part by weight) soybean hypocotylmaterial (Reference Example 1-3) Starch 20.00 Sugar 32.00 Kinako(roasted soybean powder) 10.00 Black sesame seeds 20.00 Baking powderq.s. Flavor q.s. Sweetener q.s. Total 100.00 parts by weight

Example 4-E-46 Bean Confectionery

A bean confectionery was produced under the same conditions as inExample 4-E-45, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.75 parts by weight of the solidextract prepared in Example 1-1 by evaporating Liquid Extract 1-2obtained using 75Et to dryness.

Example 4-E-47 Nama-Fu Confectionery (Fresh Gluten Cake)

A nama-fu confectionery (fresh gluten cake) was produced using thefollowing ingredients.

Equol-containing fermented 10.00 (part by weight) soybean hypocotylmaterial (Reference Example 1-3) Gluten 20.00 Rice flour 15.00 Kinako(roasted soybean powder) q.s. Emulsifier q.s. Honey q.s. Sorbitol 20.00Chocolate 30.00

Example 4-E-48 Nama-Fu Confectionery (Fresh Gluten Cake)

A nama-fu confectionery (fresh gluten cake) was produced under the sameconditions as in Example 4-E-47, except for using, instead of theequol-containing fermented soybean hypocotyl material, 1.25 parts byweight of the solid extract prepared in Example 1-1 by evaporatingLiquid Extract 1-2 obtained using 75Et to dryness.

Example 4-E-49 Wafer (Rich Type)

A wafer (rich type) was produced in accordance with a general productionmethod of a wafer, using the following ingredients.

Equol-containing fermented 0.40 (part by weight) soybean hypocotylmaterial (Reference Example 1-3) Wheat flour 100.00 Sugar 2.00 Fats andoils 3.00 Whole egg 3.00 Skimmed milk 2.00 Sodium bicarbonate 0.30 Cow'smilk 50.00 Water 100.00

Example 4-E-50 Wafer (Rich Type)

A wafer (rich type) was produced under the same conditions as in Example4-E-49, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 parts by weight of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-E-51 Wafer (Plain)

A wafer (plain) was produced in accordance with a general productionmethod of a wafer, using the following ingredients.

Equol-containing fermented 0.40 (part by weight) soybean hypocotylmaterial (Reference Example 1-3) Wheat flour 100.00 Fats and oils 2.50Whole egg 0.30 Ammonium carbonate 0.40 Sodium bicarbonate 0.30 Lecithin0.05 Water 150.00

Example 4-E-52 Wafer (Plain)

A wafer (plain) was produced under the same conditions as in Example4-E-51, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 parts by weight of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-E-53 Cheese Cracker

A cheese cracker was produced in accordance with a general productionmethod, using the following ingredients.

Equol-containing fermented 0.40 (part by weight) soybean hypocotylmaterial (Reference Example 1-3) Wheat flour 100.00 Fats and oils 9.00Malt extract 1.30 Sodium bicarbonate 0.60 Spice 0.10 Cheese powder 13.00Sugar 80.00 Table salt 0.90 Ammonium carbonate 0.60 Lecithin 0.05 Water33.00

Example 4-E-54 Cheese Cracker

A cheese cracker was produced under the same conditions as in Example4-E-53, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 parts by weight of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-E-55 Yokan (Azuki Bean Jelly)

Yokan (azuki bean jelly) was produced in accordance with a generalproduction method, using the following ingredients.

Equol-containing fermented 4.00 (part by weight) soybean hypocotylmaterial (Reference Example 1-3) Agar 7.50 Granulated sugar 320.00 Redbean paste 620.00 Starch syrup 50.00 Water 300.00

Example 4-E-56 Yokan (Azuki Bean Jelly)

Yokan (azuki bean jelly) was produced under the same conditions as inExample 4-E-55, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.5 parts by weight of the solidextract prepared in Example 1-1 by evaporating Liquid Extract 1-2obtained using 75Et to dryness.

Example 4-E-57 Soft Cookie

A soft cookie was produced in accordance with a general productionmethod, using the following ingredients.

Equol-containing fermented soybean 4.00 (part by weight) hypocotylmaterial (Reference Example 1-3) Wheat flour 200.00 Butter 150.00 Sugar80.00 Whole egg 150.00

Example 4-E-58 Soft Cookie

A soft cookie was produced under the same conditions as in Example4-E-57, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.5 parts by weight of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-E-59 Rice Cracker

A rice cracker was produced in accordance with a general productionmethod, using the following ingredients.

Equol-containing fermented soybean  0.40 (part by weight) hypocotylmaterial (Reference Example 1-3) Regular rice 100.00 Hot water q.s.

Example 4-E-60 Rice Cracker

A rice cracker was produced under the same conditions as in Example4-E-59, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.5 parts by weight of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-E-61 Suhama (Japanese Traditional Confectionery)

Suhama (Japanese traditional confectionery) was produced in accordancewith a general production method, using the following ingredients.

Equol-containing fermented soybean 4.00 (part by weight) hypocotylmaterial (Reference Example 1-3) White superior soft sugar 600.00 Kinako(roasted soybean powder) 400.00 Starch syrup 130.00 Water 180.00

Example 4-E-62 Suhama (Japanese Traditional Confectionery)

Suhama (Japanese traditional confectionery) was produced under the sameconditions as in Example 4-E-61, except for using, instead of theequol-containing fermented soybean hypocotyl material, 0.5 parts byweight of the solid extract prepared in Example 1-1 by evaporatingLiquid Extract 1-2 obtained using 75Et to dryness.

Example 4-E-63 Dry Confectionery

A dry confectionery was produced in accordance with a general productionmethod, using the following ingredients.

Equol-containing fermented soybean 4.00 (part by weight) hypocotylmaterial (Reference Example 1-3) Wasanbon sugar 500.00 Shitori (formaking the ingredients wet 25.00 during the process) Kanbaiko 75.00(ground toasted rice cake)

Example 4-E-64 Dry Confectionery

A dry confectionery was produced under the same conditions as in Example4-E-63, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.5 parts by weight of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-E-65 Fruit Gummy Candy

A fruit gummy candy was produced in accordance with a general productionmethod, using the following ingredients.

Equol-containing fermented soybean 4.00 (part by weight) hypocotylmaterial (Reference Example 1-3) Sugar 30.00 Starch syrup 50.00 Gelatin7.00 Fruit juice 5.00 Citric acid 1.50 Flavor 0.20

Example 4-E-66 Fruit Gummy Candy

A fruit gummy candy was produced under the same conditions as in Example4-E-65, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.5 parts by weight of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-E-67 Soy Flour-Containing Baked Confectionery

An amount of 1.25 wt. % of equol-containing fermented soybean hypocotylmaterial (Reference Example 1-3) and 25 wt. % of soy flour, as well asraisins, butter, sugar, egg, orange peel, pineapple, hard-to-digestdextrin, cacao mass, almond slices, cocoa powder, table salt, bakingpowder, and flavors were mixed in a suitable amount. The resultantmixture (100 wt. %) was then formed into a rectangle (about 1.5 cm×1.5cm×10 cm) using a mold to be baked in an oven at 180° C., therebyproducing a soy flour-containing baked confectionery.

Example 4-E-68 Soy Flour-Containing Baked Confectionery

A soybean flour-containing baked confectionery was produced under thesame conditions as in Example 4-E-67, except for using, instead of theequol-containing fermented soybean hypocotyl material, 0.25 parts byweight of the solid extract prepared in Example 1-1 by evaporatingLiquid Extract 1-2 obtained using 75Et to dryness.

Example 4-F Seasoning

Hereinafter, specific production examples of the seasoning according tothe present invention are given.

Example 4-F-1 Barbecue Sauce

A barbecue sauce having the following composition was produced.

Equol-containing fermented 4.00 (Kg) soybean hypocotyl material(Reference Example 1-3) Reducing starch syrup 20.00 Trehalose 0.50 Mirin4.00 Soy sauce (dark) 24.00 Apple puree 19.00 Garlic paste 4.50 Gingerpaste 4.50 Sesame oil 0.10 Xanthan gum 0.50 Red pepper powder 0.10 Blackpepper powder 0.05 Citric acid (crystalline) 0.20 Toasted sesame seeds0.40 Water Balance Total 100.00 kg

Example 4-F-2 Barbecue Sauce

A barbecue sauce was produced under the same conditions as in Example4-F-1, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.5 kg of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-F-3 Tomato Ketchup

A tomato ketchup having the following composition was produced.

Equol-containing fermented 4.00 (Kg) soybean hypocotyl material(Reference Example 1-2) Tomato paste 40.00 Wine vinegar 18.00Fructose/glucose syrup 5.00 Soy sauce (dark) 24.00 Table salt 2.50Garlic powder 0.07 Onion powder 0.10 Cinnamon powder 0.02 Clove powder0.01 Water Balance Total 100.00 kg

Example 4-F-4 Tomato Ketchup

A tomato ketchup was produced under the same conditions as in Example4-F-3, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.5 kg of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-F-5 Tsuyu (Dipping Sauce)

Tsuyu (dipping sauce for tempura, i.e. Japanese deep-fried food) havingthe following composition was produced.

Equol-containing fermented 4.00 (Kg) soybean hypocotyl material(Reference Example 1-2) Sugar 7.00 Soy sauce (dark) 26.00 Soy sauce(light) 10.00 Fish sauce 5.00 Mirin 10.00 Sodium L-glutamate 3.00monohydrate Table salt 0.60 Water Balance Total 100.00 Kg

Example 4-F-6 Tsuyu (Soup)

Tsuyu (soup) was produced under the same conditions as in Example 4-F-5,except for using, instead of the equol-containing fermented soybeanhypocotyl material, 0.5 kg of the solid extract prepared in Example 1-1by evaporating Liquid Extract 1-2 obtained using 75Et to dryness.

Example 4-F-7 Dressing

The aqueous phase and the oil phase of the following composition weremixed in a weight ratio of 7:3 to produce a dressing (separate type).

Composition of Aqueous Phase Equol-containing fermented 4.00 (Kg)soybean hypocotyl material (Reference Example 1-3) Sugar 6.00 Soy sauce(dark) 20.00 Brewed vinegar (acidity: 10%) 8.50 Fish sauce 3.00 Lemonjuice 2.00 Table salt 2.00 Sodium L-glutamate monohydrate 0.20 Redpepper powder 0.05 Toasted sesame seeds 0.30 Xanthan gum 0.10 WaterBalance Total 100.00 kg Composition of Oil Phase Sesame oil 20.00 (Kg)Corn salad oil 79.00 Seasoning flavor 1.00 Total 100.00 kg

Example 4-F-8 Dressing

A dressing (separate type) was produced under the same conditions as inExample 4-F-7, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.5 kg of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-F-9 Sesame Dipping Sauce for Shabu-Shabu (Thinly Sliced Meetand Vegetables Boiled Quickly in Hot Water)

Sesame dipping sauce for shabu-shabu having the following compositionwas produced.

Equol-containing fermented 0.40 (Kg) soybean hypocotyl material(Reference Example 1-2) Soy sauce (light) 12.00 Brewed vinegar 11.00Garlic powder 0.10 Ginger powder 0.10 White pepper 0.10 Chicken extract3.00 Yeast extract 3.00 Sesame oil 1.00 Fructose/glucose syrup 8.00Mirin 15.00 White miso 10.00 Thickener 3.50 Sugar 8.00 Table salt 10.00Flavor 0.15 Water Balance Total 100.00 kg

Example 4-F-10 Sesame Dipping Sauce for Shabu-Shabu (Thinly Sliced Meetand Vegetables Boiled Quickly in Hot Water)

Sesame dipping sauce for shabu-shabu was produced under the sameconditions as in Example 4-F-9, except for using, instead of theequol-containing fermented soybean hypocotyl material, 0.05 kg of thesolid extract prepared in Example 1-1 by evaporating Liquid Extract 1-2obtained using 75Et to dryness.

Example 4-F-11 Barbecue Sauce (Miso Flavor)

Barbecue sauce (miso flavor) having the following composition wasproduced.

Equol-containing fermented 4.00 (Kg) soybean hypocotyl material(Reference Example 1-3) Soy sauce (dark) 25.00 Miso 14.00 D-sorbitol12.00 Table salt 3.20 Garlic powder 3.30 Garlic paste 2.00 Beef extract1.00 Toasted sesame seeds 0.50 Sodium L-glutamate 0.50 monohydrateGinger paste 0.40 Amino acid seasoning 0.01 Thickener 3.50 Sugar 10.00Water Balance Total 100.00 kg

Example 4-F-12 Barbecue Sauce (Miso Flavor)

Barbecue sauce (miso flavor) was produced under the same conditions asin Example 4-F-11, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.05 kg of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-F-13 Snack Seasoning

A snack seasoning having the following composition was produced.

Equol-containing fermented soybean 0.40 (part by weight) hypocotylmaterial (Reference Example 1-3) Table salt 14.00 Tricalcium phosphate3.00 Disodium succinate 0.20 Sodium L-glutamate monohydrate 7.00Powdered soy sauce 3.00 Yeast extract 1.50 Onion powder 1.50 Paprikapowder 1.50 Garlic powder 6.00 Red pepper powder 0.50 Chicken consommé21.00 Beef consommé 4.00 Stevia 0.60 Glucose 13.00 Spice 0.01 Cornstarch23.00 Sugar 2.00

Example 4-F-14 Snack Seasoning

A snack seasoning was produced under the same conditions as in Example4-F-13, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 parts by weight of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-F-15 Emulsified Soy Sauce

Emulsified soy sauce having the following composition was produced.

Equol-containing fermented 0.40 (part by weight) soybean hypocotylmaterial (Reference Example 1-2) Salad oil 34.00 Soy sauce (dark) 53.00Gum Arabic 1.00 Sugar 12.00

Example 4-F-16 Emulsified Soy Sauce

Emulsified soy sauce was produced under the same conditions as inExample 4-F-15, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.05 parts by weight of the solidextract prepared in Example 1-1 by evaporating Liquid Extract 1-2obtained using 75Et to dryness.

Example 4-F-17 Dressing

A dressing (emulsified type) having the following composition wasproduced.

Equol-containing fermented 0.40 (part by weight) soybean hypocotylmaterial (Reference Example 1-2) Salad oil 35.00 Vinegar 10.00 Sugar11.00 Table salt 4.00 Xanthan gum 0.30 Water Balance Total 100.00 partsby weight

Example 4-F-18 Dressing

A dressing (emulsified type) was produced under the same conditions asin Example 4-F-17, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.05 parts by weight of the solidextract prepared in Example 1-1 by evaporating Liquid Extract 1-2obtained using 75Et to dryness.

Example 4-F-19 Sauce for Kabayaki (Broiled Seafood)

Sauce for Kabayaki (broiled seafood) having the following compositionwas produced.

Equol-containing fermented soybean 0.40 (part by weight) hypocotylmaterial (Reference Example 1-3) D-sorbitol 10.00 Thickener 0.30 Soysauce (dark) 38.00 Mirin 20.00 Umami extract 2.00 Table salt 1.80 SodiumL-glutamate monohydrate 1.45 Sodium inosinate 0.025 Sodium guanylate0.025 Trehalose 15.00 Sugar 6.00 Caramel coloring 0.50 Onion color 0.10Flavor 0.50 Water Balance Total 100.00 parts by weight

Example 4-F-20 Sauce for Kabayaki (Broiled Seafood)

Sauce for Kabayaki (broiled seafood) was produced under the sameconditions as in Example 4-F-19, except for using, instead of theequol-containing fermented soybean hypocotyl material, 0.05 parts byweight of the solid extract prepared in Example 1-1 by evaporatingLiquid Extract 1-2 obtained using 75Et to dryness.

Example 4-F-21 Seasoning for Squid Delicacies

A Seasoning for squid delicacies having the following composition wasproduced.

Equol-containing fermented soybean 0.40 (part by weight) hypocotylmaterial (Reference Example 1-2) Soy sauce (dark) 20.00 Soy sauce(light) 10.00 Fish sauce 5.00 Mirin 10.00 Brewed vinegar 2.00 Sugar38.00 Table salt 1.00 Sodium L-glutamate 2.00 monohydrate Umami extract1.40 Disodium succinate 0.20 Garlic powder 0.05 Water Balance Total100.00 parts by weight

Example 4-F-22 Seasoning for Squid Delicacies

A Seasoning for squid delicacies was produced under the same conditionsas in Example 4-F-21, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.05 parts by weight of the solidextract prepared in Example 1-1 by evaporating Liquid Extract 1-2obtained using 75Et to dryness.

Example 4-G Retort-Packed Food

Hereinafter, specific production examples of the retort-packed foodaccording to the present invention are given.

Example 4-G-1 Retort-Packed Curry

Retort-packed curry was produced in accordance with a general productionmethod, using the following composition.

Equol-containing fermented soybean hypocotyl 0.40 (Kg) material(Reference Example 1-3) Curry powder 1.80 Onion 7.00 Unsalted butter4.50 Garlic paste 0.60 Ginger paste 0.50 Wheat flour 4.50 Fruit chutney3.00 Tomato paste 0.50 Table salt 0.55 Sodium L-glutamate monohydrate0.48 Nucleic acid seasoning 0.015 Demi-glace flavor 1.00 Pork extractseasoning 0.50 Bouillon flavor 1.00 Potato 7.00 Carrot 5.00 Beef 10.00Water Balance Total 100.00 Kg

Example 4-G-2 Retort-Packed Curry

Retort-packed curry was produced under the same conditions as in Example4-G-1, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 kg of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-G-3 Retort-Packed Premix of Chinese-Style Donburi

Retort-packed Premix of Chinese-style Donburi was produced in accordancewith a general production method, using the following composition.

Equol-containing fermented soybean hypocotyl 0.40 (Kg) material(Reference Example 1-3) Soy sauce (light) 72.00 Sake 46.00 Table salt4.00 Sodium L-glutamate monohydrate 1.30 Nucleic acid seasoning 0.07Chicken extract powder 1.00 Sugar 7.00 Water 209.00 Pork 200.00 Squid120.00 Shrimp 80.00 Shiitake (Lentinus edodes) 70.00 Boiled bambooshoots 160.00 Carrot 60.00 Onion 240.00

Example 4-G-4 Retort-Packed Premix of Chinese-Style Donburi

Retort-packed premix of Chinese-style donburi was produced under thesame conditions as in Example 4-G-3, except for using, instead of theequol-containing fermented soybean hypocotyl material, 0.05 kg of thesolid extract prepared in Example 1-1 by evaporating Liquid Extract 1-2obtained using 75Et to dryness.

Example 8 Processed Meat Product

Hereinafter, specific production examples of the processed meat productaccording to the present invention are given.

Example 4-H-1 Sausage

A sausage was produced in accordance with a general production method ofa sausage, using the ingredients of the following composition.

Meat portion 5 mm-minced pork (foreshank) 70.00 (Kg) 5 mm-minced pork(lard) 10.00 Ice water 20.00 Total 100.00 kg Auxiliary ingredientsEquol-containing fermented soybean hypocotyl 0.40 (Kg) material(Reference Example 1-3) Table salt 1.70 Sodium nitrite 0.012Polyphosphate 0.30 Sodium L-ascorbate 1.00 Sodium casein 0.50 Whitepepper powder 0.10 Potassium sorbate 0.20 Amino acid seasoning 0.30Spice mix 0.50

Example 4-H-2 Sausage

A sausage was produced under the same conditions as in Example 4-H-1,except for using, instead of the equol-containing fermented soybeanhypocotyl material, 0.05 kg of the solid extract prepared in Example 1-1by evaporating Liquid Extract 1-2 obtained using 75Et to dryness.

Example 4-H-3 Smoked Sausage

The meat portion and the seasoning having the following componentsrespectively were mixed at a ratio of 16:1. Using the resultant mixture,smoked sausages were produced in accordance with a general productionmethod of a smoked sausage.

Meat portion Minced pork (foreshank) 85.00 (Kg) Whole egg 4.00 Cow'smilk 3.00 Ice water 7.00 Seasoning Equol-containing fermented soybeanhypocotyl 0.40 (Kg) material (Reference Example 1-3) Table salt 24.00Amino acid seasoning 3.20 Spice 8.65 Minced Onion 16.00 Sugar 48.00

Example 4-H-4 Smoked Sausage

A smoked sausage was produced under the same conditions as in Example4-H-3, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 kg of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-H-5 Hamburger Steak

A hamburger steak was produced in accordance with a general productionmethod, using the following composition.

Equol-containing fermented soybean 0.40 (part by weight) hypocotylmaterial (Reference Example 1-3) Mixture of ground beef and pork 45.00Pork 9.00 Onion 12.00 Whole egg 5.00 Granulated soy protein 9.00 Sodiumcasein 5.00 Carrageenan 1.50 Locust bean gum 0.50 Spice 0.30 Yeastextract 0.20 Table salt 1.00 Sugar 1.20 Water-soluble dietary fiber 1.00Water Balance Total 100.00 parts by weight

Example 4-H-6 Hamburger Steak

A hamburger steak was produced under the same conditions as in Example4-H-5, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 parts by weight of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-I Fish Paste

Hereinafter, specific production examples of the fish paste according tothe present invention are given.

Example 4-I-1 Fried Kamaboko (Fish Paste Cake)

A fried Kamaboko (fish paste cake) was produced in accordance with ageneral production method of a fish paste cake, using the ingredients ofthe following composition.

Equol-containing fermented soybean hypocotyl 0.40 (Kg) material(Reference Example 1-3) Surimi 50.00 Table salt 1.50 Amino acidseasoning 0.30 Glycine 0.50 Sodium acetate anhydrous 0.30 Potato starch8.00 Mirin 1.00 Ice water Balance Total 100.00 Kg

Example 4-I-2 Fried Kamaboko (Fish Paste Cake)

A fried kamaboko (fish paste cake) was produced under the sameconditions as in Example 4-I-1, except for using, instead of theequol-containing fermented soybean hypocotyl material, 0.05 kg of thesolid extract prepared in Example 1-1 by evaporating Liquid Extract 1-2obtained using 75Et to dryness.

Example 4-J Processed Egg Product

Hereinafter, specific production examples of the processed egg productaccording to the present invention are given.

Example 4-J-1 Thick Japanese Omelet

A thick Japanese omelet was produced in accordance with a generalproduction method of a thick Japanese omelet, using the ingredients ofthe following composition.

Equol-containing fermented soybean 0.40 (part by weight) hypocotylmaterial (Reference Example 1-3) Whole egg 70.00 Table salt 0.40 Starch2.00 Soup stock 2.00 Sugar 1.20 Sodium acetate 1.00 Water Balance Total100.00 parts by weight

Example 4-J-2 Thick Japanese Omelet

A thick Japanese omelet was produced under the same conditions as inExample 4-J-1, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.05 parts by weight of the solidextract prepared in Example 1-1 by evaporating Liquid Extract 1-2obtained using 75Et to dryness.

Example 4-K Canned Food

Hereinafter, specific production examples of the canned food accordingto the present invention are given.

Example 4-K-1 Canned Mandarin Orange

A canned mandarin orange was produced by filling mandarin orange pulpand a syrup having the following composition in a can

Equol-containing fermented soybean 0.40 (part by weight) hypocotylmaterial (Reference Example 1-3) Sugar 10.00 Fructose/glucose syrup10.00 Citric acid 0.25 Trisodium citrate 0.10 Water Balance Total 100.00parts by weight

Example 4-K-2 Canned Mandarin Orange

Canned mandarin orange was produced under the same conditions as inExample 4-K-1, except for using, instead of the equol-containingfermented soybean hypocotyl material, 0.05 parts by weight of the solidextract prepared in Example 1-1 by evaporating Liquid Extract 1-2obtained using 75Et to dryness.

Example 4-L Bread Product

Hereinafter, specific production examples of the bread according to thepresent invention are given.

Example 4-L-1 Loaf of Bread

A loaf of bread was produced using dough having the followingcomposition.

Sponge Dough Strong flour 70.00 (Kg) Yeast food 0.10 Live yeast 2.00Water 40.00 Main Dough Equol-containing fermented soybean hypocotyl 0.40(Kg) material (Reference Example 1-3) Strong flour 30.00 Sugar 3.00Table salt 2.00 Skimmed milk 2.00 Shortening 5.00 Water 25.00

Example 4-L-2 Loaf of Bread

A loaf of bread was produced under the same conditions as in Example4-L-1, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 kg of the solid extract prepared inExample 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-M Frozen Desert

Hereinafter, specific production examples of the frozen dessertaccording to the present invention are given.

Example 4-M-1 Ice Cream

Ice cream having the following composition was produced.

Equol-containing fermented soybean 0.40 (part by weight) hypocotylmaterial (Reference Example 1-3) Raw cream 8.80 Butter 5.00 Starch syrup16.00 Skimmed milk 6.50 Trehalose 7.00 Sugar 5.00 Stabilizer 0.20 Flavor0.15 Water Balance Total 100.00 parts by weight

Example 4-M-2 Ice Cream

Ice cream was produced under the same conditions as in Example 4-M-1,except for using, instead of the equol-containing fermented soybeanhypocotyl material, 0.05 parts by weight of the solid extract preparedin Example 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-N Processed Soybean Food

Hereinafter, specific production examples of the processed soybean foodaccording to the present invention are given.

Example 4-N-1 Fermented Soybeans

An amount of 99.6 parts by weight of steamed soybeans and 0.4 parts byweight of equol-containing fermented soybean hypocotyl material(Reference Example 1-3) were mixed. The resultant mixture was theninoculated with Bacillus natto for fermentation; thereby fermentedsoybeans were produced.

Example 4-N-2 Fermented Soybeans

Fermented Soybeans were produced under the same conditions as in Example4-N-1, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 parts by weight of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

Example 4-N-3 Tofu

Tofu having the following composition was produced.

Equol-containing fermented soybean 0.40 (part by weight) hypocotylmaterial (Reference Example 1-3) Soya Milk 99.30 Magnesium chloride 0.20Potassium sorbate 0.10 Total 100.00 parts by weight

Example 4-N-4 Tofu

Tofu was produced under the same conditions as in Example 4-N-3, exceptfor using, instead of the equol-containing fermented soybean hypocotylmaterial, 0.05 parts by weight of the solid extract prepared in Example1-1 by evaporating Liquid Extract 1-2 obtained using 75Et to dryness.

Example 4-O-1 Cooked Rice Product

Hereinafter, specific production examples of the cooked rice productaccording to the present invention are given.

Example 4-O-1 Rice Gruel

Rice gruel was produced in accordance with a general production methodof rice gruel, using the ingredients of the following composition.

Equol-containing fermented soybean 0.40 (part by weight) hypocotylmaterial (Reference Example 1-3) Rice 160.00 Table salt 1.00 Water900.00

Example 4-O-2 Rice Gruel

Rice gruel was produced under the same conditions as in Example 4-O-1,except for using, instead of the equol-containing fermented soybeanhypocotyl material, 0.05 parts by weight of the solid extract preparedin Example 1-1 by evaporating Liquid Extract 1-2 obtained using 75Et todryness.

Example 4-O-3 Steamed Glutinous Rice with Chestnuts

Steamed glutinous rice with chestnuts was produced in accordance with ageneral production method, using the following ingredients.

Equol-containing fermented soybean 4.00 (part by weight) hypocotylmaterial (Reference Example 1-3) Glutinous rice 1000.00 Peeled chestnut300.00 Soup stock 230.00 Black sesame seeds 30.00 Salt solution 6.00

Example 4-O-4 Steamed Glutinous Rice with Chestnuts

Steamed glutinous rice with chestnuts was produced under the sameconditions as in Example 4-O-3 except for using, instead of theequol-containing fermented soybean hypocotyl material, 0.5 parts byweight of the solid extract prepared in Example 1-1 by evaporatingLiquid Extract 1-2 obtained using 75Et to dryness.

Example 4-P Soup

Hereinafter, specific production examples of the soup according to thepresent invention are given.

Example 4-P-1 Corn Cream Soup

Corn cream soup was produced in accordance with a general productionmethod of a soup, using the ingredients of the following composition.

Equol-containing fermented soybean 0.40 (part by weight) hypocotylmaterial (Reference Example 1-3) Cow's milk 180.00 Corn 190.00 Consomme20.00 Salad oil 15.00 Table salt q.s. Pepper q.s. Water 360.00

Example 4-P-2 Corn Cream Soup

Corn cream soup was produced under the same conditions as in Example4-P-1, except for using, instead of the equol-containing fermentedsoybean hypocotyl material, 0.05 parts by weight of the solid extractprepared in Example 1-1 by evaporating Liquid Extract 1-2 obtained using75Et to dryness.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 is an electropherogram showing the total proteins contained inequol-containing fermented soybean hypocotyl obtained in ReferenceExample 1-1, soybean cotyledon, and the soybean hypocotyl.

FIG. 2 is an electropherogram showing the results of the detection ofmajor allergens (Gym4, Gm30K, and Gm28K) contained in theequol-containing fermented soybean hypocotyl obtained in ReferenceExample 1-1, soybean cotyledon, and the soybean hypocotyl.

FIG. 3 is an electropherogram showing the results of the detection ofmajor allergens (7S globulin mix, oleocine, and trypsin inhibitor)contained in the equol-containing fermented soybean hypocotyl obtainedin Reference Example 1-1, soybean cotyledon, and the soybean hypocotyl.

FIG. 4 shows TLC analysis results indicating the equol contents ofLiquid Extracts 1-2 and 2-2 in Example 1-1.

FIG. 5 shows TLC analysis results indicating the saponin contents ofLiquid Extracts 1-2 and 2-2 in Example 1-1.

FIG. 6 shows the TLC analysis results indicating the equol contents ofLiquid Extracts 1-2 and Residue 2 in Example 1-1.

FIG. 7 shows TLC analysis results indicating the saponin contents ofLiquid Extracts 1-2 and Residue 2 in Example 1-1.

FIG. 8 shows TLC analysis results of solutions eluted by silica gelcolumn chromatography in Example 2-1.

The invention claimed is:
 1. A method for producing an equol-containingextract comprising: Step I-1 of subjecting an equol-containing fermentedsoybean to extraction using an aqueous ethanol solution containingbetween 65-90% ethanol as an extractant, to thereby produce a firstliquid extract and a first residue, and collecting said first liquidextract; and Step I-2 of removing the aqueous ethanol extractant fromsaid first liquid extract to thereby produce a second residue,conducting extraction on said second residue using absolute ethanol asan extractant, and collecting the liquid resulting from said extractionon said second residue, wherein the saponin content in theequol-containing extract is reduced.
 2. The method for producing anequol-containing extract according to claim 1, wherein theequol-containing fermented soybean is obtained by fermenting a soybeanwith an equol-producing microorganism.
 3. The method according to claim1, which further comprises: Step I-3 of removing a solvent from saidliquid collected in Step I-2, suspending the resulting residue inhexane, and then collecting the insoluble residue; Step I-4 ofsubjecting the insoluble residue obtained in Step I-3 to extractionusing a mixture of hexane and ether as an extractant, and thencollecting the resulting liquid extract; and Step I-5 of subjecting theliquid extract obtained in Step I-4 to silica gel column chromatography,and obtaining an equol-containing fraction.
 4. The method according toclaim 1, wherein the equol-containing extract is in a form suitable forfood.
 5. The method according to claim 1, wherein the equol-containingextract contains glycitein compounds.
 6. A method for producing anequol-containing extract comprising: Step I-1 of subjecting anequol-containing fermented soybean to extraction using an aqueousethanol solution as an extractant, to thereby produce a first liquidextract and a first residue, and collecting said first liquid extract;Step I-2 of removing the aqueous ethanol extractant from said firstliquid extract to thereby produce a second residue, conductingextraction on said second residue using ethanol as an extractant, andcollecting the liquid resulting from said extraction on said secondresidue; Step I-3 of removing a solvent from said liquid collected inStep I-2, suspending the resulting residue in hexane, and thencollecting the insoluble residue; Step I-4 of subjecting the insolubleresidue obtained in Step I-3 to extraction using a mixture of hexane andether as an extractant, and then collecting the resulting liquidextract; and Step I-5 of subjecting the liquid extract obtained in StepI-4 to silica gel column chromatography, and obtaining anequol-containing fraction.
 7. The method for producing anequol-containing extract according to claim 6, wherein the ethanolconcentration of the aqueous ethanol solution used in Step I-1 is 20 to98 vol. %.
 8. The method for producing an equol-containing extractaccording to claim 6, wherein the equol-containing fermented soybean isobtained by fermenting a soybean with an equol-producing microorganism.9. The method according to claim 6, wherein the equol-containing extractis in a form suitable for food.
 10. The method according to claim 6,wherein the equol-containing extract contains glycitein compounds.